Inhibitors of cacna1a/alpha1a subunit internal ribosomal entry site (ires) and methods of treating spinocerebellar ataxia type 6

ABSTRACT

The invention provides methods of treating polyglutamine diseases, e.g., spinocerebellar ataxia Type 6, in a subject, comprising administering to the subject an IRES inhibitor in an amount effective for treating the SCA6 in the subject. Also provided herein are the IRES inhibitors, and pharmaceutical compositions comprising the same.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/450,760, filed Jun. 24, 2019, now U.S. Pat. No. 11,034,962, which isa divisional of U.S. application Ser. No. 15/743,560, filed Jan. 10,2018, which is a U.S. national stage application of InternationalApplication No. PCT/US16/45492, filed Aug. 4, 2016, which claimspriority to U.S. Provisional Patent Application No. 62/200,933, filedAug. 4, 2015, the invention of which is incorporated herein by referencein their entirety.

GRANT FUNDING

This invention was made with government support under Grant No.R01NS082788 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: 843,341 byte ACII (Text) file named“27373/49857A seqlisting.txt,” created on Aug. 2, 2016.

BACKGROUND

Spinocerebellar ataxia type 6 (SCA6), a form of spinocerebellar ataxia(SCA), is a dominantly-inherited neurodegenerative disease characterizedby progressive ataxia and Purkinje cell degeneration, associated withCAG repeat expansions in the gene, CACNA1A. SCA6 is a severeneurological disorder, and one of the most common SCAs worldwide(Moseley et al., Neurology. 1998; 51(6):1666-171; Wu et al., Clin Genet.2004; 65(3):209-14; Jayadev et al., J Neurol Sci. 2006; 250(1-2):110-3;Basri et al., J Hum Genet. 2007; 52(10):848-55; Klockgether. Cerebellum.2008; 7(2):101-5; Wardle et al., J Neurol. 2009; 256(3):343-348),roughly as prevalent as amyotrophic lateral sclerosis (Kurtzke J F. AdvNeurol. 1982; 36:281-302; Zhuchenko et al., Nature Genetics. 1997;15(1):62-9; Craig K, et al., Ann Neurol. 2004; 55(5):752-5).

Initially, patients with SCA6 experience problems with coordination andbalance (ataxia). Other early signs and symptoms of SCA6 include speechdifficulties, involuntary eye movements (nystagmus), and double vision.Over time, individuals with SCA6 may develop loss of coordination intheir arms, tremors, and uncontrolled muscle tensing (dystonia). Signsand symptoms of SCA6 typically begin in a person's forties or fiftiesbut can appear anytime from childhood to late adulthood. Most peoplewith this disorder require wheelchair assistance by the time they are intheir sixties. Patients with genetically distinct forms of SCA,including SCA6, become disabled and may progress to severeincapacitation. Many patients die prematurely due to aspirationpneumonia or respiratory failure (Perlman S L. Handb Clin Neurol. 2011;100:113-40; Klockgether T., Curr Opin Neurol. 2011; 24(4):339-45;Marelli C, et al., Rev Neurol (Paris). 2011; 167(5):385-400). The adventof modern molecular genetics has enabled the confirmed moleculardiagnosis and characterization of many distinct forms of SCA (Durr A.Lancet Neurol. 2010; 9(9):885-94). The most reliable prevalenceestimates of these heterogeneous disorders are on the order of18-50/100,000 (Craig et al., (2004), supra, Tsuji S, et al., Cerebellum.2008; 7(2):189-97). Thus, these disorders create a substantial economicand societal burden (Lopez-Bastida et al., Mov Disord. 2008;23(2):212-7).

Current treatment of patients with SCA6 is focused on the treatment ofmanifestations. For example, acetazolamide is given to patients toeliminate episodes of ataxia, while vestibular suppressants are given toreduce vertigo and/or osscilopsia. Ophthalmology consultation isprovided for refractive or surgical management of diplopia. Clonazepamis given for REM sleep disorders. Home modifications are suggested forsafety and convenience. Canes, walking sticks, and walkers areprescribed in order to prevent falling. Physical therapy may also beprescribed to maximize compensation and strength, while speech therapyand communication devices are given for dysarthria. Weighted eatingutensils and dressing hooks are suggested, while video esophagrams areprovided to identify safest behaviors and consistency of food leastlikely to trigger aspiration. Feeding assessment when dysphagia becomestroublesome is considered. Furthermore, weight control, as obesityexacerbates ambulation and mobility difficulties, is provided. Moreover,CPAP may be administered to patients for sleep apnea.

However, no preventive treatment exists for the numerous polyglutamine(polyQ) diseases, including SCA6, and, currently, there are notreatments that target SCA6 itself. Thus, a method of treating SCA6,rather than a method of treating SCA6 manifestations, is needed.

SUMMARY

Presented herein are data relating to the origin and function of theα1ACT polypeptide in physiology and disease. Previously, it has beendemonstrated that α1ACT is generated from the full-length α1A transcriptby means of a cellular internal ribosomal entry site (IRES) locatedwithin the α1A mRNA, i.e., that the CACNA1A gene is bi-cistronic (Du etal., Cell 154:118-133 (2013). The α1ACT protein containing the normalpolyQ tract is a transcription factor that binds and enhances expressionof several Purkinje cell (PC)-expressed genes, promotes neuriteoutgrowth, and partially rescues the CACNA1A knockout phenotype. α1ACTwith an expanded polyQ has altered function, reduces viability of cellsin vitro, and causes gait impairment and cerebellar cortical atrophy invivo. A truly bi-cistronic, dual-function, cellular gene encoding twoproteins with completely distinct functions, in this case an ion channeland a transcription factor, was reported (Du et al. (2013), supra). Thisgene expression strategy demonstrates a novel role for an IRES incoordinating gene expression, as well as a potential therapeutic targetfor disease modifying therapy.

Provided herein are data demonstrating the selective inhibition of theexpression of α1ACT encoded by the CACNA1A gene, without inhibiting theexpression of α1A encoded by the same gene, upon administration of anIRES inhibitor described herein. Also provided herein are datademonstrating the inhibition of the translational initiation of CACNA1Agene-driven α1ACT by eIF4AII and EIF4GII. Further provided herein aredata demonstrating the inhibition or prevention of Purkinje celldegeneration caused by CACNA1A gene-driven α1ACT_(SCA6). Furthermore,provided herein are data demonstrating the the inhibition or preventionof ataxia and motor deficits caused by CACNA1A gene-driven α1ACT_(SCA6).Without being bound to a particular theory, these data support thatadministration of an IRES inhibitor described herein achieves treatmentof SCA6 and other related diseases described herein.

Accordingly, the invention provides a method of treating a geneticdisease in a subject. The method comprises the step of administering tothe subject an IRES inhibitor in an amount effective for treating thegenetic disease. In exemplary embodiments, the genetic disease is atrinucleotide repeat disorder. In exemplary embodiments, thetrinucleotide repeat disorder is a polyglutamine disease. In exemplaryaspects, the polyglutamine disease is spinocerebellar ataxia Type 6(SCA6). In exemplary aspects, the IRES inhibitor of the invention areantisense molecules, as described herein. In exemplary aspects, the IRESinhibitor is a small, non-coding RNA, or an antisense nucleic acidanalog thereof. In exemplary aspects, the antisense molecule is amicroRNA (miRNA) or small interfering RNA (siRNA). In exemplary aspects,the antisense molecule binds to the IRES of the CACNA1A gene and,optionally, to Argonaute 4 (Ago4). In exemplary aspects, the antisensemolecule binds to a portion of the IRES comprising the sequence of SEQID NO: 180. In exemplary aspects, the antisense molecule binds to aportion of the sequence of SEQ ID NO: 180. In exemplary aspects, theantisense molecule inhibits the expression of α1ACT encoded by theCACNA1A gene. In exemplary aspects, the antisense molecule does notinhibit expression of α1A encoded by the CACNA1A gene. In exemplaryaspects, the antisense molecule selectively inhibits the expression ofα1ACT encoded by the CACNA1A gene, but does not inhibit expression ofα1A encoded by the CACNA1A gene. In exemplary embodiments, the IRESinhibitor is (i) an antisense molecule that binds to a portion of anIRES of a CACNA1A gene comprising the sequence of SEQ ID NO: 180,optionally, wherein the antisense molecule binds to Argonaute 4 (Ago4),(ii) a vector encoding the antisense molecule, (iii) a cell comprisingthe vector or antisense molecule, (iv) a extracellular vesiclecomprising the antisense molecule, or (v) a combination thereof.Optionally, the vector is a recombinant expression vector of asdescribed herein, the cell is a cell as described herein, or theextracellular vesicle is an extracellular vesicle as described herein.

Accordingly, the invention provides a method of treating spinocerebellarataxia Type 6 (SCA6). In exemplary embodiments, the method comprises thestep of administering to the subject an IRES inhibitor in an amounteffective for treating the SCA6 in the subject. In exemplaryembodiments, the method comprises administering to the subject (i) anantisense molecule that binds to a portion of an IRES of a CACNA1A genecomprising the sequence of SEQ ID NO: 180, optionally, wherein theantisense molecule binds to Argonaute 4 (Ago4), (ii) a vector encodingthe antisense molecule, (iii) a cell comprising the vector or antisensemolecule, (iv) a extracellular vesicle comprising the antisensemolecule, or (v) a combination thereof. Optionally, the vector is arecombinant expression vector of as described herein, the cell is a cellas described herein, or the extracellular vesicle is an extracellularvesicle as described herein.

The invention also provides a method of treating a subject with apredisposition to spinocerebellar ataxia Type 6 (SCA6). In exemplaryembodiments, the method comprises the step of administering to thesubject an IRES inhibitor in amount effective for delaying developmentof SCA6 in the subject. In exemplary embodiments, the method comprisesadministering to the subject (i) an antisense molecule that binds to aportion of an IRES of a CACNA1A gene comprising the sequence of SEQ IDNO: 180, optionally, wherein the antisense molecule binds to Argonaute 4(Ago4), (ii) a vector encoding the antisense molecule, (iii) a cellcomprising the vector or antisense molecule, (iv) a extracellularvesicle comprising the antisense molecule, or (v) a combination thereof.Optionally, the vector is a recombinant expression vector of asdescribed herein, the cell is a cell as described herein, or theextracellular vesicle is an extracellular vesicle as described herein.

IRES inhibitors, including (i) antisense molecules that bind to aportion of an IRES of a CACNA1A gene comprising the sequence of SEQ IDNO: 180, optionally, wherein the antisense molecule binds to Argonaute 4(Ago4), (ii) a vector encoding the antisense molecule, (iii) a cellcomprising the vector or antisense molecule, (iv) a extracellularvesicle comprising the antisense molecule, or (v) a combination thereof,are provided herein. Pharmaceutical compositions comprising an IRESinhibitor, including (i) antisense molecules that bind to a portion ofan IRES of a CACNA1A gene comprising the sequence of SEQ ID NO: 180,optionally, wherein the antisense molecule binds to Argonaute 4 (Ago4),(ii) a vector encoding the antisense molecule, (iii) a cell comprisingthe vector or antisense molecule, (iv) a extracellular vesiclecomprising the antisense molecule, or (v) a combination thereof, areprovided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1K evidence that miR-3191-5p, in collaboration with Ago4,inhibits the IRES-driven translation of α1ACT_(SCA6) while sparingα1AscA6 expression and α1AscA6 mRNA levels in HEK293 cells. (FIG. 1A)miR-711, -3191-5p, and -4786-3p significantly decreased CACNA1AIRES-driven luciferase activities. (FIG. 1B) miR-3191-5p down-regulatedα1ACT-FLAG expression but spared α1A-FLAG expression, although miR-711and -4′786-3p did both. (FIG. 1C) miR-3191-5p produced no significantchange in α1A-Q33 mRNA levels. (FIG. 1D) Nucleotide sequence of themiR-3191-5p binding region of the CACNA1A IRES and mutated CACNA1A IRES(IRESmut) templates bearing C>G and G>C substitutions within thepredicted miR-3191-5p binding site. (FIG. 1E) Dual luciferase IRESmutreporter activities were not inhibited by miR-3191-5p. (FIG. 1F)CACNA1A-FLAG vector with IRESmut still expressed α1ACT, which was notinhibited by miR-3191-5p. (FIG. 1G) miR-3191-5p did not affect theexpression levels of endogenous α1A mRNA in HEK293 cells. (FIG. 1H, FIG.1I) The knockdown of Ago4 reversed the down-regulating effects ofmiR-3191-5p on the CACNA1A IRES-driven luciferase activities (FIG. 1H)and translation of α1ACT (FIG. 1I). (FIG. 1J, FIG. 1K) The levels ofmiR-3191-5p (FIG. 1J) and α1A-Q33 mRNA (FIG. 1K) precipitated byAgo4-specific antibodies were greater than control IgG. Data aremeans±S.E.M. **P<0.01, ***P<0.001. n.s.: not significant; siC: siRNAcontrol; NC: miRNA negative control.

FIGS. 2A-2H demonstrate that the complex of eIF4AII and eIF4GII directlyact on CACNA1A IRES to enhance IRES-driven translation of α1ACT that isinhibited by miR-3191-5p bound to Ago4 in HEK293 cells. (FIG. 2A)eIF4AII and eIF4GII increased CACNA1A IRES-driven luciferase activitiesthat were reversed by the treatment with miR-3191-5p. (FIG. 2B)miR-3191-5p reversed the up-regulating effects of eIF4AII and eIF4GII onα1ACT-FLAG expression. (FIG. 2C) eIF4AII and eIF4GII did not affect theα1A mRNA levels. (FIG. 2D, FIG. 2E) miR-3191-5p did not reverse theup-regulating effects of eIF4AII or eIF4GII on CACNA1A IRESmut-drivenluciferase activities (FIG. 2D) and on the translation of α1ACTexpressed from CACNA1A IRESmut vectors (FIG. 2E). (FIG. 2F)Immunoprecipitation from transfected HEK293 cells with vectorsexpressing eIF4AII and eIF4GII. (FIG. 2G) The knockdown of eIF4GIIshowed greater silencing effects on the CACNA1A IRES-driven luciferaseactivities than eIF4AII. (FIG. 2H) Both eIF4AII and eIF4GII bound to themRNA transcribed from IRESstopmut-α1ACT vectors that were inhibited bythe treatment with miR-3191-5p. Data are means±S.E.M. *P<0.05, **P<0.01,***P<0.001. n.s.: not significant; C: control vector; AI: eIF4AI; AII:eIF4AII; GI: eIF4GI; GII: eIF4GII; IgG: control IgG; siC: siRNA control;NC: miRNA negative control; miR-C: miR-control expressing vector;miR-3191: miR-3191-5p expressing vector. Input, 10%.

FIGS. 3A-3H demonstrate the physiological and pathological effects ofAAV-mediated delivery of either α1ACT_(WT) or α1ACT_(SCA6) into the WTneonatal mice. (FIG. 3A) The co-immunostaining using GFP-specific orFLAG-specific antibodies with staining for calbindin, a Purkinje cellmarker. (FIG. 3B) Representative images of immunofluorescence. (FIG. 3Cto FIG. 3E) Histopathological features of the cerebellum of miceinjected with AAV. The molecular layer thickness (FIG. 3C), the densityof dendritic tree (FIG. 3D), and the Purkinje cell count (FIG. 3E)(n=6). (FIG. 3F to FIG. 3H) The clinical symptoms of mice injected withAAV. The body weight (FIG. 3F), rotarod task (FIG. 3G), open field assay(FIG. 3H) (n=12; 6 male and 6 female mice per each group). Data aremeans±S.E.M. **P<0.01 and ***P<0.001. n.s.: not significant. Scale bars:(FIG. 3A) and (FIG. 3B), 100 μm.

FIGS. 4A-4G evidence the therapeutic effects of treatment with the viraladministration of miR-3191-5p on the mouse phenotypes induced byAAV-mediated delivery of α1ACT_(SCA6). (FIG. 4A) Representative imagesof immunofluorescence. (FIG. 4B to FIG. 4D) Histopathological featuresof the cerebellum of mice injected with AAV. The molecular layerthickness (FIG. 4B), the density of dendritic tree (FIG. 4C), and thePurkinje cell count (FIG. 4D) (n=6). (FIG. 4E to FIG. 4G) The clinicalsymptoms of mice injected with AAV. The body weight (FIG. 4E), rotarodtask (FIG. 4F), open field assay (FIG. 4G) (n=12; 6 male and 6 femalemice per each group). WT-GFP: WT-GFP mice; WT-Q33-mock: WT-Q33-miR-mockmice; WT-Q33-3191: WT-Q33-miR-3191-5p mice. Data are means±S.E.M.**P<0.01 and ***P<0.001. n.s.: not significant. Scale bars: (A), 100 μm.

FIGS. 5A-5C evidence the stem-loop structure of CACNA1A IRES and thepredicted binding site of miR-711, -3191-5p, and -4′786-3p. (FIG. 5A)The CentroidFold program demonstrated the stem-loop structure of CACNA1AIRES. (FIG. 5B) The miRNA_Targets program showed the predicted bindingsites of miR-711, -3191-5p, and -4′786-3p. (FIG. 5C) miR-711, -3191-5p,and -4786-3p target the region within CACNA1A IRES between 185 bp and534 bp upstream from the estimated translational initiation site ofα1ACT where we have previously identified an important role in theIRES-driven translation of α1ACT (9).

FIG. 6 demonstrates the preparation of a bicistronic CACNA1A IRESreporter vector. Schematic representations of a bicistronic controlreporter vector (pRF) and a bicistronic CACNA1A IRES reporter vector(pRF-IRES). SV40: simian virus 40; polyA: polyadenylation signalsequence.

FIGS. 7A-7B evidence that miR-3191-5p inhibits the IRES-driventranslation of α1ACT_(WT) while sparing α1A_(WT) expression and α1A_(WT)mRNA levels, while miR-711 and -4786 down-regulate α1A_(WT) protein andmRNA in HEK293 cells. (FIG. 7A) Western blot analysis showed thatmiR-3191-5p down-regulated α1ACT-Q11-FLAG expression but sparedα1A-Q11-FLAG expression, although miR-711 and -4′786-3p did both. (FIG.7B) miR-711 and -4′786-3p down-regulated α1A-Q11 mRNA levels byapproximately half relative to a negative control. miR-3191-5p, however,produced no significant change in α1A-Q11 mRNA levels. Data aremeans±S.E.M. ***P<0.001. n.s.: not significant; NC: miRNA negativecontrol.

FIGS. 8A-8B evidence the preparation of mutated CACNA1A IRES (IRESmut)templates bearing C>G and G>C substitutions within the predictedmiR-3191-5p binding site. (FIG. 8A, FIG. 8B) Schematic representationsof the original CACNA1A IRES (FIG. 8A) and mutated CACNA1A IRES(IRESmut) templates (FIG. 8B). C>G and G>C substitutions within thepredicted miR-3191-5p binding site are shown in lowercases. We alsoperformed C>G and G>C substitutions within the complementary bindingsite of the sequence targeted by miR-3191-5p, shown in lowercases, tomaintain the structure of CACNA1A IRESmut same as that of the original.

FIG. 9 demonstrates that Ago4 preferentially binds to CACNA1A IRES. Wealso performed an immunoprecipitation-coupled qRT-PCR using abicistronic control (pRF) or CACNA1A IRES reporter vector (pRF-IRES) inHEK293 cells. Compared to the case of pRF vector, Ago4-specificantibodies preferentially precipitated mRNA transcribed from pRF-IRESvector than control IgG. Data are means±S.E.M. **P<0.01. n.s.: notsignificant.

FIGS. 10A-10B demonstrate the preparation of truncated transgenes ofCACNA1A second cistron that lack the sequence of 5′ up-stream fromCACNA1A IRES (IRESstopmut-α1ACT). (FIG. 10A) Schematic representation oftruncated transgenes of CACNA1A second cistron that lack the sequence of5′ up-stream from CACNA1A IRES (IRESstopmut-α1ACT). (FIG. 10B) Westernblot analysis showed that the IRESstopmut-α1ACT vectors expressedα1ACT-Q11-FLAG or α1ACT-Q33-FLAG but not full-length α1A-Q11-FLAG orα1A-Q33-FLAG. CACNA1A-Q11, -Q33: vectors expressing CACNA1A-encodedFLAG-tagged peptides (α1A-FLAG and α1ACT-FLAG) with 11Q, -33Q.

FIGS. 11A-11C demonstrate the widespread viral transduction throughoutthe brain and cerebellum of WT mice injected with AAV. (FIG. 11A)Schematic representation of the AAV vectors carrying the truncatedtransgenes of CACNA1A second cistron that lack the sequence of 5′up-stream from CACNA1A IRES and express either α1ACT-Q11 or -Q33 taggedwith a C-terminal FLAG epitope under the control of CACNA1A IRES. (FIG.11B) Western blot analysis showed the strong expression of GFP,α1ACT-Q11-FLAG, or α1ACT-Q33-FLAG in the brain and cerebellum of WT miceinjected with AAV. The continuous expression of GFP, α1ACT-Q11-FLAG, orα1ACT-Q33-FLAG was observed in AAV injected mice at 4 and 12 weeks ofage. (FIG. 11C) The expression levels of α1ACT mRNA transcribed from thetransgenes delivered by AAV-α1ACT-Q11 or AAV-α1ACT-Q33 into thecerebellum. ITR: inverted terminal repeats; SV40: simian virus 40;polyA: polyadenylation signal sequence; WT-GFP: WT-GFP mice;WT-α1ACT-Q11: WT-α1ACT-Q11 mice; WT-α1ACT-Q33: WT-α1ACT-Q33 mice. Dataare means±S.E.M. n.s.: not significant.

FIGS. 12A-12F demonstrate the video-assisted computerized treadmill forthe gait analysis of WT mice injected with AAV. (FIG. 12A to FIG. 12F)The Digigait assay revealed that WT-α1ACT-Q33 mice exhibited shorterstride length (FIG. 12A, FIG. 12C, FIG. 12E) and greater stridefrequencies (FIG. 12B, FIG. 12D, FIG. 12F) compared to WT-GFP mice andWT-α1ACT-Q11 mice from 4 weeks old (n=12; 6 male and 6 female mice pereach group). GFP: WT-GFP mice; Q11: WT-α1ACT-Q11 mice; Q33: WT-α1ACT-Q33mice; L fore: Left forelimb; R fore: Right forelimb; L hind: Lefthindlimb; R hind: Right hindlimb. Data are means±S.E.M. **P<0.01.

FIGS. 13A-13B evidence the widespread viral transduction throughout thebrain and cerebellum of WT mice injected with AAV. (FIG. 13A) Westernblot analysis revealed the persistent expression of α1ACT-Q33-FLAG orGFP in the brain and cerebellum of WT mice co-injected withAAV-α1ACT-Q33 and either AAV-miR-mock or AAV-miR-3191-5p, or injectedwith AAV-GFP for at least 12 weeks. We found the significantly decreasedlevels of α1ACT-Q33-FLAG expression in the brain and cerebellum ofWT-Q33-miR-3191-5p mice as compared to WT-Q33-miR-mock mice. (FIG. 13B)qRT-PCR studies of total RNA showed that the α1ACT-Q33 mRNA levels inthe cerebellum of WT-Q33-miR-3191-5p mice were comparable with those ofWT-Q33-miR-mock mice. WT-GFP: WT-GFP mice; WT-Q33-mock: WT-Q33-miR-mockmice; WT-Q33-3191: WT-Q33-miR-3191-5p mice. Data are means±S.E.M. n.s.:not significant.

FIGS. 14A-14F demonstrate the video-assisted computerized treadmill forthe gait analysis of mice injected with AAV. (FIG. 14A to FIG. 14F) TheDigigait assay revealed that the treatment with AAV-miR-3191-5pameliorated the disease phenotypes of shorter stride length (FIG. 14A,FIG. 14C, FIG. 14E) and greater stride frequencies (FIG. 14B, FIG. 14D,FIG. 14F) in WT-Q33-miR-3191-5p mice. GFP: WT-GFP mice; Q33-mock:WT-Q33-miR-mock mice; Q33-3191: WT-Q33-miR-3191-5p mice; L fore: Leftforelimb; R fore: Right forelimb; L hind: Left hindlimb; R hind: Righthindlimb. (n=12; 6 male and 6 female mice per each group). Data aremeans±S.E.M. **P<0.01.

FIGS. 15A-15F demonstrate a histopathological examination of the brain,cerebellum, heart, lung, liver, and kidney of WT mice treated withAAV-miR-3191-5p. (FIG. 15A to FIG. 15F) Histopathological examinationrevealed that no obvious morphological anomalies in the brain (FIG.15A), cerebellum (FIG. 15B), heart (FIG. 15C), lung (FIG. 15D), liver(FIG. 15E), and kidney (FIG. 15F) of the 12 week-old WT mice injectedwith AAV-miR-3191-5p (WT-miR-3191-5p) compared to age-matched WT mice(WT). Scale bars: (FIG. 15A), 500 μm; and (FIG. 15B) to (F), 200 μm.

FIGS. 16A-16H provide data demonstrating that somatic gene transfer ofCACNA1A IRES-driven α1ACTSCA6 causes Purkinje cell degeneration in mice.(FIG. 16A) Representative immunofluorescence images of AAV9-injectedmouse brain and cerebellum from 4-week-old mice stained withFLAG-specific antibodies and fluorescent secondary antibody. Scale bar,5 mm. (FIG. 16B) Relative α1ACT-FLAG immunofluorescence intensities inAAV9-injected mouse hippocampus, cerebral cortex, and cerebellum (n=6).Relative α1ACT-FLAG immunofluorescence is expressed as signalintensities per unit area quantified by the National Institutes ofHealth (NIH) ImageJ software. n.s., not significant. (FIG. 16C) Relativeα1ACT mRNA expression in the cerebellum of AAV9-injected mice at 4 weeksof age (n=4). (FIG. 16D) Western blot and densitometric analyses showingα1ACT-Q11-FLAG and α1ACT-Q33-FLAG expression in the cerebellum ofAAV9-injected mice at 4 weeks of age (n=4). (FIG. 16E) Representativeimmunofluorescence images of AAV9-injected mouse cerebellum from4-week-old mice stained with calbindin-specific antibodies andfluorescent secondary antibody. Scale bar, 100 μm. (FIG. 16F to FIG.16H) Molecular layer thickness (FIG. 16F), density of dendritic tree(FIG. 16G), and Purkinje cell count of AAV9-injected mouse cerebellumfrom 4-week-old mice (n=6) (FIG. 16H). GFP, AAV9-GFP mice; Q11,AAV9-α1ACT-Q11 mice; Q33, AAV9-α1ACT-Q33 mice. All data representmeans±SEM. **P<0.01. Student's t test in (B) to (D). One-way analysis ofvariance (ANOVA) in (FIG. 16F) to (FIG. 16H).

FIGS. 17A-17E provide data demonstrating that CACNA1A IRES-drivenα1ACTSCA6 causes an early-onset ataxia and motor deficits in mice. (FIG.17A to FIG. 17E) Clinical features of AAV9-injected mice. Body weight(FIG. 17A), rotarod test (FIG. 17B), and open-field assay (FIG. 17C) at4 weeks of age. Stride length (FIG. 17D) and stride frequencies (FIG.17E) assessed by DigiGait analysis at 4 weeks of age (n=12, 6 male and 6female mice per group). GFP, AAV9-GFP mice; Q11, AAV9-α1ACT-Q11 mice;Q33, AAV9-α1ACT-Q33 mice. All data represent means±SEM. *P<0.05,**P<0.01. Two-way ANOVA in (FIG. 17A) and (FIG. 17B). One-way ANOVA in(FIG. 17C) to (FIG. 17E).

FIGS. 18A-18G provide data demonstrating that miR-3191-5p inhibits theCACNA1A IRES-driven translation of α1ACT while sparing α1A and CACNA1AmRNA expression in HEK293 cells. (FIG. 18A) Schematic representation ofa bicistronic control reporter vector and a bicistronic CACNA1A IRESreporter vector. SV40, simian virus 40; polyA, polyadenylation signalsequence. (FIG. 18B) Relative dual-luciferase CACNA1A IRES reporteractivities in HEK293 cells treated with miR-711, miR-3191-5p,miR-4786-3p, or an miRNA negative control (NC) (n=6). The ratio offirefly luciferase to Renilla luciferase activities of a bicistronicCACNA1A IRES reporter vector was normalized to that of a bicistroniccontrol reporter vector. (FIG. 18C and FIG. 18D) Western blot anddensitometric analyses showing α1A-FLAG and α1ACT-FLAG expression inHEK293 cells treated with three miRNAs or NC (n=6). (FIG.18C)CACNA1A-Q11. (FIG. 18D) CACNA1A-Q33. GAPDH,glyceraldehyde-3-phosphate dehydrogenase. (FIG. 18E and FIG. 18F)Relative CACNA1A-Q11 (FIG. 18E) and CACNA1A-Q33 (FIG. 18F) mRNAexpression in HEK293 cells treated with three miRNAs or NC (n=6). (FIG.18G) Relative endogenous CACNA1A mRNA expression in HEK293 cells treatedwith miR-3191-5p or NC (n=6). All data represent means±SEM. **P<0.01,***P<0.001. Student's t test in (FIG. 18B) to (FIG. 18G).

FIGS. 19A-19E provide data demonstrating that Ago4 is required formiR-3191-5p-mediated inhibition of CACNA1A IRES-driven α1ACTtranslation. (FIG. 19A) Relative dual-luciferase CACNA1A IRES reporteractivities in HEK293 cells treated with si-Ago1, si-Ago2, si-Ago3,si-Ago4, or a scrambled control siRNA (si-C) in the presence ofmiR-3191-5p or an miRNA NC (n=6). The ratio of firefly luciferase toRenilla luciferase activities of a bicistronic CACNA1A IRES reportervector was normalized to that of a bicistronic control reporter vector.(FIG. 19B) Western blot and densitometric analyses showing α1A-Q33-FLAGand α1ACT-Q33-FLAG expression in HEK293 cells treated with four siRNAsor si-C in the presence of miR-3191-5p or NC (n=6). (FIG. 19C and FIG.19D) Amount of miR-3191-5p (FIG. 19C) and CACNA1A-Q33 (FIG. 19D) mRNAbinding to Ago1 to Ago4 that was immunoprecipitated using antibodiesagainst Ago1 to Ago4 or a control immunoglobulin G (IgG) (n=6). (FIG.19E) Relative amount of endogenous miR-3191-5p in HEK293 cells treatedwith si-Ago4 or si-C(n=6). All data represent means±SEM. **P<0.01,***P<0.001. Student's t test in (FIG. 19A) to (FIG. 19E).

FIGS. 20A-20L provide data demonstrating that miR-3191-5p inhibits thetranslational initiation of CACNA1A IRES-driven α1ACT by eIF4AII andeIF4GII. (FIG. 20A to FIG. 20D) Relative dual-luciferase CACNA1A IRESreporter activities in HEK293 cells treated by overexpression of eithereIF4AI (AI) (A), eIF4AII (AII) (FIG. 20B), eIF4GI (GI) (FIG. 20C),eIF4GII (GII) (FIG. 20D), or a control vector (FIG. 20C) in the presenceof miR-3191-5p or an miRNA NC (n=6). The ratio of firefly luciferase toRenilla luciferase activities of a bicistronic CACNA1A IRES reportervector was normalized to that of a bicistronic control reporter vector.(FIG. 20E and FIG. 20F) Western blot and densitometric analyses showingα1A-Q33-FLAG and α1ACT-Q33-FLAG expression in HEK293 cells treated byoverexpression of either AII (FIG. 20E), GII (FIG. 20F), or C in thepresence of miR-3191-5p or NC (n=6). (FIG. 20G and FIG. 20H) RelativeCACNA1A-Q33 mRNA expression in HEK293 cells treated by overexpression ofeither AII (FIG. 20G), GII (FIG. 20H), or C in the presence ofmiR-3191-5p or NC (n=6). (FIG. 20I and FIG. 20J) Amount ofIRES-α1ACT-Q33 mRNA binding to AII (FIG. 20I), GII (FIG. 20J), or acontrol IgG in the presence of vectors expressing miR-3191-5p (miR-3191)or an miRNA control (miR-C) that was immunoprecipitated using antibodiesagainst AII, GII, or a control IgG (n=6). (FIG. 20K) Immunoprecipitation(IP) from HEK293 cells transfected with AII- and GII-expressing vectorsusing an antibody against AII, GII, or a control IgG. Recovered proteinswere analyzed using Western blot. Input, 10%. (FIG. 20L) Relativedual-luciferase CACNA1A IRES reporter activities in HEK293 cells treatedwith si-AII, si-GII, or a scrambled si-C(n=6). The ratio of fireflyluciferase to Renilla luciferase activities of a bicistronic CACNA1AIRES reporter vector was normalized to that of a bicistronic controlreporter vector. All data represent means±SEM. *P<0.05, **P<0.01,***P<0.001. Student's t test in (FIG. 20A) to (FIG. 20F), (FIG. 20I),(FIG. 20J), and (FIG. 20L). One-way ANOVA in (FIG. 20G) and (FIG. 20H).

FIGS. 21A-21H data demonstrating that AAV9-mediated therapeutic deliveryof miR-3191-5p blocks CACNA1A IRES-driven α1ACT translation in mice.(FIG. 21A and FIG. 21B) Representative immunofluorescence images ofAAV9-injected mouse brain and cerebellum from 4-week-old mice stainedwith GFP-specific (FIG. 21A) and FLAG-specific (FIG. 21B) antibodies andfluorescent secondary antibody. Scale bar, 5 mm. (FIG. 21C and FIG. 21D)Relative GFP (FIG. 21C) and α1ACT-FLAG (FIG. 21D) immunofluorescenceintensities in AAV9-injected mouse hippocampus, cerebral cortex, andcerebellum (n=6). Relative GFP and relative FLAG immunofluorescence areexpressed as signal intensities per unit area quantified by NIH ImageJsoftware. (FIG. 21E) Relative α1ACT mRNA expression in the cerebellum ofAAV9-injected mice at 4 weeks of age (n=4). (FIG. 21F) Western blot anddensitometric analyses showing α1ACT-Q33-FLAG expression in thecerebellum of AAV9-injected mice at 4 weeks of age (n=4). (FIG. 21G andFIG. 21H) Amount of miR-3191-5p (FIG. 21G) and α1ACT-Q33 (FIG. 21H) mRNAbinding to Ago4 that was immunoprecipitated using an antibody againstAgo4 or a control IgG in the cerebellum of AAV9-Q33-miR-3191-5p mice at4 weeks of age (n=6). GFP, AAV9-GFP mice; mock, AAV9-Q33-miR-mock mice;3191, AAV9-Q33-miR-3191-5p mice. All data represent means±SEM. **P<0.01.One-way ANOVA in (FIG. 21C). Student's t test in (FIG. 21D) to (FIG.21H).

FIGS. 22A-22D provide data demonstrating that miR-3191-5p preventsPurkinje cell degeneration caused by CACNA1A IRES-driven α1ACTSCA6 inmice. (FIG. 22A) Representative immunofluorescence images ofAAV9-injected mouse cerebellum from 4-week-old mice stained withcalbindin-specific antibodies and fluorescent secondary antibody. Scalebar, 100 μm. (FIG. 22B to FIG. 22D) Molecular layer thickness (FIG.22B), density of dendritic tree (FIG. 22C), and Purkinje cell counts(FIG. 22D) in AAV9-injected mouse cerebellum from 4-week-old mice (n=6).GFP, AAV9-GFP mice; mock, AAV9-Q33-miR-mock mice; 3191,AAV9-Q33-miR-3191-5p mice. All data represent means±SEM. **P<0.01.Student's t test in (FIG. 22B) to (FIG. 22D).

FIGS. 23A-23E provide data demonstrating that AAV9-miR-3191-5p preventsthe ataxia and motor deficits caused by CACNA1A IRES-driven α1ACTSCA6 inmice. (FIG. 23A to FIG. 23E) Clinical features of AAV9-injected mice.Body weight (FIG. 23A), rotarod test (FIG. 23B), and open-field assay(FIG. 23C) at 4 weeks of age. Stride length (FIG. 23D) and stridefrequencies (FIG. 23E) assessed by DigiGait analysis at 4 weeks of age(n=12, 6 male and 6 female mice per group). GFP, AAV9-GFP mice; mock,AAV9-Q33-miR-mock mice; 3191, AAV9-Q33-miR-3191-5p mice. All datarepresent means±SEM. *P<0.05, **P<0.01. Two-way ANOVA in (FIG. 23A) and(FIG. 23B). Student's t test in (FIG. 23C) to (FIG. 23E).

FIGS. 24A-24D include a schematic representation of AAV9 vectors andAAV9 transduction efficiency into Purkinje cells. (FIG. 24A) Schematicrepresentation of truncated transgenes of CACNA1A second cistron thatlack the sequence of 5′ upstream from CACNA1A IRES (IRES-α1ACT). Thesequence of nt 4962 to 7757 of full-length CACNA1A cDNA (NM_001127222.1)corresponds to that of CACNA1A IRES and α1ACT open reading frame. Thesequence of nt 5013 to 5015 is modified from “TAT” to “TAG” of stopcodon. (FIG. 24B) Schematic representations of the AAV9 vectorsexpressing either CACNA1A IRES-driven α1ACT-Q11 or CACNA1A IRES-drivenα1ACT-Q33 tagged with a C-terminal FLAG epitope. ITR: inverted terminalrepeats; WPRE: woodchuck hepatitis virus posttranscriptional regulatoryelement; SV40: simian virus 40; polyA: polyadenylation signal sequence.(FIG. 24C) Representative immunofluorescence images of AAV9-GFP-injectedmouse cerebellum from 4-week-old mice co-stained with calbindin-specificand GFP-specific antibodies and fluorescent secondary antibody (scalebars: 100 μm). Phosphate saline buffer (PBS)-injected mice were used ascontrols. (FIG. 24D) Purkinje cell count of AAV9-injected mousecerebellum relative to PBS-injected mouse cerebellum (n=6). 75.4±8.9% ofcalbindin positive cells were positive for GFP staining in thecerebellum of AAV9-GFP mice. Calbindin(+): calbindin positive cells.GFP(+): GFP positive cells.

FIGS. 25A-25B provide data demonstrating a histopathological examinationof the AAV9-injected mouse cerebral cortex and hippocampus. (FIG. 25A,FIG. 25B) Representative images of (FIG. 25A) cerebral cortex and (FIG.25B) hippocampus of AAV9-GFP mice, AAV9-α1ACT-Q11mice, andAAV9-α1ACT-Q33 mice stained with Hematoxylin and eosin. Scale bars: 500μm in (FIG. 25A) and (FIG. 25B).

FIGS. 26A-26H demonstrate the long-term follow-up of AAV9-injected mousebehavioral phenotypes and CACNA1A IRES-driven α1ACT expression in mice.(FIG. 26A to FIG. 26F) Clinical features of AAV9-injected mice. (FIG.26A) Rotarod test and (FIG. 26B) open field assay, (FIG. 26C) stridelength and (FIG. 26D) stride frequencies assessed by Digigait analysisat 12 weeks of age. (FIG. 26E) Rotarod test and (FIG. 26F) open fieldassay at 30 weeks of age (n=12:6 male and 6 female mice per each group).(FIG. 26G) Western blot and densitometric analyses (FIG. 26H) showingα1ACT-FLAG expression in the cerebellum of AAV9-injected mice at 30weeks of age (n=4). GFP: AAV9-GFP mice; Q11: AAV9-α1ACT-Q11 mice; Q33:AAV9-α1ACT-Q33 mice. All data represent mean±SEM. *P<0.05, **P<0.01.Two-way ANOVA in (FIG. 26A) and (FIG. 26E). One-way ANOVA in (FIG. 26B)to (FIG. 26D), and (FIG. 26F). Student's t-test in (FIG. 26G).

FIGS. 27A-27D demonstrate the stem-loop structure of CACNA1A IRES andthe predicted binding sites of miR-711, miR-3191-5p, and miR-4786-3p.(FIG. 27A) Secondary structure of stem-loop of CACNA1A IRES calculatedby the CentroidFold. (B to D) The nucleotide sequence of binding sitesof (FIG. 27B) miR-711, (FIG. 27C) miR-3191-5p, and (FIG. 27D)miR-4786-3p within CACNA1A IRES predicted by the miRNA_Targets program.

FIGS. 28A-28E provide data demonstrating the effects of miR-3191-5p onmutated CACNA1A IRES templates. (FIG. 28A, FIG. 28B) Schematicrepresentations of (FIG. 28A) the original CACNA1A IRES and (FIG. 28B)mutated CACNA1A IRES (CACNA1A IRESmut) templates. C>G and G>Csubstitutions within the predicted miR-3191-5p binding site are shown inlowercase. We also performed C>G and G>C substitutions within thecomplementary binding site of the sequence targeted by miR-3191-5p, alsoshown in lowercase, to maintain the stem-loop structure of CACNA1AIRESmut as same as that of the original. (FIG. 28C) Nucleotide sequenceof the miR-3191-5p binding site within CACNA1A IRES and CACNA1A IRESmuttemplates. (FIG. 28D) Relative dual luciferase CACNA1A IRESmut reporteractivities in HEK293 cells treated with miR-3191-5p or a miRNA negativecontrol (NC) (n=6). The ratio of firefly luciferase to Renillaluciferase activities of a bicistronic CACNA1A IRESmut reporter vectorwas normalized to that of a bicistronic control reporter vector. (FIG.28E) Western blot and densitometric analyses showing α1A-Q33-FLAG andα1ACT-Q33-FLAG expression from CACNA1A IRESmut vectors in HEK293 cellstreated with miR-3191-5p or NC (n=6). All data represent mean±SEM. n.s.:not significant. Student's t-test in (FIG. 28D) and (FIG. 28E).

FIGS. 29A-29F provide data demonstrating the effects of eIF4AII andeIF4GII on mutated CACNA1A IRES templates in the presence or absence ofmiR-3191-5p. (FIG. 29A, FIG. 29B) Relative dual luciferase CACNA1AIRESmut reporter activities in HEK293 cells treated with over-expressionof either (FIG. 29A) eIF4AII (AII), (FIG. 29B) eIF4GII (GII), or acontrol vector (FIG. 29C) in the presence of miR-3191-5p or a miRNAnegative control (NC) (n=6). The ratio of firefly luciferase to Renillaluciferase activities of a bicistronic CACNA1A IRESmut reporter vectorwas normalized to that of abicistronic control reporter vector. (FIG.29C, FIG. 29D) Western blot and densitometric analyses showingα1A-Q33-FLAG and α1ACT-Q33-FLAG expression from CACNA1A IRESmut vectorsin HEK293 cells treated with over-expression of either (FIG. 29C) AII,(FIG. 29D) GII, or C in the presence of miR-3191-5p or NC (n=6). (FIG.29E) Schematic diagram of the effect of eIF4AII and eIF4GII on CACNA1AIRES-driven α1ACT translation. The complex of eIF4AII and eIF4GIIdirectly acts on CACNA1A IRES to enhance the initiation of IRES-drivenα1ACT translation. (FIG. 29F) Schematic diagram of the inhibitory effectof miR-3191-5p bound to Ago4. miR-3191-5p, in collaboration with Ago4,binds to CACNA1A IRES to inhibit the interaction between CACNA1A IRESand the complex of eIF4AII and eIF4GII. All data represent mean±SEM.**P<0.01, ***P<0.001. n.s.:not significant. Student's t-test in (FIG.29A) to (FIG. 29D).

FIGS. 30A-30C provide data demonstrating cotransduction ofAAV9-α1ACT-Q33 with either AAV9-miR-mock or AAV9-miR-3191-5p. (FIG. 30A)Schematic representations of the AAV9 vectors expressing GFP with eithermiR-3191-5p or a scrambled miR (miR-mock). ITR: inverted terminalrepats; WPRE: woodchuck hepatitis virus posttranscriptional regulatoryelement; SV40: simian virus 40; polyA: polyadenylation signal sequence.(FIG. 30B) Representative immunofluorescence images of AAV9-injectedmouse cerebellum from 4-week-old mice stained with GFP- andFLAG-specific antibodies and fluorescent secondary antibody (scale bars:100 μm). (FIG. 30C) The ratio of Purkinje cells stained with both FLAG-and GFP-specific antibodies (both FLAG and GFP positive cells) to thosewith FLAG-specific antibody (FLAG positive cells). FLAG positive cellswere transduced with AAV9-α1ACT-Q33. GFP positive cells were transducedwith either AAV9-miR-mock or AAV9-miR-3191-5p (n=6). AAV9-GFP: AAV9-GFPmice; AAV9-Q33-miRmock: AAV9-Q33-miR-mock mice; AAV9-Q33-miR-3191-5p:AAV9-Q33-miR-3191-5p mice. All data represent mean±SEM. n.s.: notsignificant. Student's t-test in (FIG. 30C).

FIGS. 31A-31G demonstrate the long-term follow-up of therapeutic effectof miR-3191-5p on mouse behavioral phenotypes and CACNA1A IRES-drivenα1ACTSCA6 expression in mice. (FIG. 31A to FIG. 31F) Clinical featuresof AAV9-injected mice. (A) Rotarod test and (FIG. 31B) open field assay,(FIG. 31C) stride length and (FIG. 31D) stride frequencies assessed byDigigait analysis at 12 weeks of age. (FIG. 31E) Rotarod test and (FIG.31F) open field assay at 30 weeks of age (n=12:6 male and 6 female miceper each group). (FIG. 31G) Western blot and densitometric analysesshowing α1ACT-Q33-FLAG expression in the cerebellum of AAV9-injectedmice at 30 weeks of age (n=4). GFP: AAV9-GFP mice; mock:AAV9-Q33-miR-mock mice; 3191: AAV9-Q33-miR-3191-5p mice. All datarepresent mean±SEM. *P<0.05, **P<0.01. Two-way ANOVA in (FIG. 31A) and(FIG. 31E). Student's t-test in (FIG. 31B) to (FIG. 31D), (FIG. 31F),and (FIG. 31G).

FIGS. 32A-32H demonstrate the potential mouse mRNAs targeted by humanmiR-3191-5p. (FIG. 32A) The list of 13 human genes of which 3′UTR hasbinding sites targeted by hsa-miR-3191-5p predicted by theTargetScanHuman 7.0 (http://www.targetscan.org/vert_70/). Among these, 7genes (PRX, ZNF781, C22orf46, ZNF23, ZNF286A, ERBB4, PTBP1) havehsa-miR-3191-5p binding sites within 3′UTR of their conserved mouseorthologs. (FIG. 32B to FIG. 32H) Relative expression of the mouse (FIG.32B) Prx, (FIG. 32C) Zfp781, (FIG. 32D) 4930407I10Rik, (FIG. 32E)Zfp612, (FIG. 32F) Zfp286, (FIG. 32G) Erbb4, and (FIG. 32H) Ptbpl mRNAsin the cerebellum of AAV9-injected mice (n=4). N/A: not assigned; WT,mock: wildtype mice injected with AAV9-miR-mock; WT, 3191: wild-typemice injected with AAV9-miR-3191-5p. All data represent mean±SEM.*P<0.05. n.s.: not significant. Student's t-test in (FIG. 32B) to (FIG.32H).

FIGS. 33A-33F demonstrate a histopathological examination of the brain,cerebellum, heart, lung, liver, and kidney of wild-type mice injectedwith AAV9-miR-3191-5p. (FIG. 33A to FIG. 33F) Histopathologicalexamination of the (FIG. 33A) brain, (FIG. 33B) cerebellum, (FIG. 33C)heart, (FIG. 33D) lung, (FIG. 33E) liver, and (FIG. 33F) kidney of the12-week-old wild-type mice injected with AAV-miR3191-5p (WT-miR-3191-5p)compared to age-matched wild-type mice (WT). All were stained withHematoxylin and eosin. Scale bars: (FIG. 33A), 500 μm; and (FIG. 33B) to(FIG. 33F), 200 μm.

DETAILED DESCRIPTION

Spinocerebellar ataxia Type 6 (SCA6)

SCA6 is a neurodegenerative disease (polyQ disorders) caused byexpansion of a polyglutamine (polyQ) tract. In SCA6, the polyQ tractexpansion is encoded by the 47^(th) exon of the CACNA1A gene. Inexemplary aspects, the normal range of polyQ tracts is 4-18, whereas thepathological range of polyQ tracts is 19-33.

The principal gene product of CACNA1A is the α1A subunit of the P/Q-typevoltage-gated Ca²⁺ channel. Voltage-gated calcium channel genes encode alarge family of channel proteins (α1 subunits) that play critical rolesin neuronal excitability, transmitter release, muscle contractility andgene expression (Catterall, Cold Spring Harbor Perspect. Biol. 3,a003947 (2011)). Genetic defects of these channels have been implicatedin a variety of neurological, cardiac and skeletal muscle disorders(Cain and Snutch, Biofactors 37: 197-205 (2011)). Diverse mutations inthe α1A subunit, CACNA1A gene, causing either loss or gain of P/Q-typechannel function, have been associated with dominantly inheritedconditions of migraine, epilepsy, and episodic and progressive ataxia(Rajakulendran et al., Nat. Rev. Neurol. 8: 86-96 (2012)). CACNA1Amutations of several types, leading to both loss and gain of channelfunction, are responsible for several types of neurological diseases,including episodic ataxia type 2, familial hemiplegic migraine, andepilepsy (Jen J, et al., Neurology. 2004; 62:17-22; Spacey S D, MaterekL A, Szczygielski B I, Bird T D. 2005; 62(2):314-6; Roubertie A, et al.,J Neurol. 2008; 255(10):1600-2).

The presence of the CAG repeat encoding a polyQ tract in the C terminusof the α1A subunit led to the obvious hypothesis that the polyQexpansion in SCA6 caused a pathological disturbance of P/Q channelfunction (Kordasiewicz H B, et al., 2007; 4(2):285-94; Lory P, et al.,IDrugs. 2010; 13(7):467-71). However, several in vitro and in vivoexpression studies have failed to demonstrate a consistent effect onchannel function (Matsuyama Z, et al., J. Neurosci. 1999; RC14:1-5;Restituito S, et al., J. Neurosci. 2000; 20(17):6394-403; Tom S, et al.,Journal of Biological Chemistry. 2000; 275(15):10893-8; Piedras-RenteriaE S, et al., J Neurosci. 2001; 21(23):9185-93; Chen H, Piedras-RenteriaE S. 2007; 292(3):C1078-86). In particular, two separate studies usingCACNA1A knock-in mice with SCA6 repeat expansions failed to demonstrateany change in P/Q channel gating properties in cerebellar neurons(Saegusa H, et al., Mol Cell Neurosci. 2007; 34(2):261-70; Watase K, etal., Proc Natl Acad Sci USA. 2008; 105(33):11987-92). Therefore, it isunlikely that SCA6 is a “channelopathy” in the classical sense. ThatSCA6 was attributed to expansion of polyQ tracts added furthercomplexity to modeling both channel function and disease pathogenesis(Zhuchenko et al., Nat. Genet. 15:62-69 (1997)).

Evidence provided herein suggests that the disease is attributable toexpression of a polyQ repeat expansion within a second CACNA1A geneproduct, α1ACT, that normally serves as a transcription factor (TF)critical for cerebellar cortical development. α1ACT is a C-terminalpeptide encoded by the α1A mRNA. SCA6-sized polyQ expansions in theα1ACT TF interrupt its cellular and molecular function, and severalstudies have shown toxicity of α1ACT with SCA6-sized polyQ expansions inmodels. Several laboratories have shown that α1ACT, which contains thepolyQ tract, is present as a stable fragment in cultured cells orcerebellar tissues (Scott V E, et al., J Neurosci. 1998; 18(2):641-7;Kubodera et al. Neurosci Lett. 2003; 341(1):74-8; Kordasiewicz et al.,Hum Mol Genet. 2006; 15(10):1587-99; Marqueze-Pouey et al., Traffic.2008; 9(7):1088-100; Ishiguro et al., Acta Neuropathol. 2010;119(4):447-64). This fragment is enriched in cerebellar nuclei,translocated based on nuclear localization signals in the α1ACT sequence(Kordasiewicz et al., supra). Finally, several groups have shown thatthe α1ACT fragment bearing SCA6-expanded polyQs, unlike the full-lengthα1A subunit, is toxic to cultured cells or primary neurons (Kubodera etal. Neurosci Lett. 2003; 341(1):74-8; Kordasiewicz et al., Hum MolGenet. 2006; 15(10):1587-99; Marqueze-Pouey et al., Traffic. 2008;9(7):1088-100; Ishiguro et al., Acta Neuropathol. 2010; 119(4):447-64).

As shown herein, α1ACT arises from a gene regulatory mechanism, that isnovel for the CACNA1A gene and for ion channel genes in general, inwhich expression of α1ACT is under the control of a cryptic cellularinternal ribosomal entry site (IRES) within the CACNA1A gene codingregion. Based on the data herein, it is hypothesized that the cellularIRES-regulated α1ACT is required for Purkinje cell development, and thatthe polyQ-expanded variant, α1ACT_(SCA6), leads to neurodegeneration.

Based at least in part on the data presented herein, the inventionprovides a method of treating SCA6 in a subject in need thereof. Inexemplary embodiments, the method comprises the step of administering tothe subject an IRES inhibitor in an amount effective for treating theSCA6 in the subject.

The invention also provides a method of treating a subject with apredisposition to spinocerebellar ataxia Type 6 (SCA6). In exemplaryembodiments, the method comprises the step of administering to thesubject an IRES inhibitor in amount effective for delaying developmentof SCA6 in the subject. In exemplary aspects, the subject with apredisposition to SCA6 is a subject who has a family history of SCA6. Inexemplary aspects, the subject with a predisposition to SCA6 is asubject who has a parent suffering from SCA6. In exemplary aspects, thesubject with a predisposition to SCA6 is a subject who has one copy ofthe altered CACNA1A gene having a number of polyQ tracts in thepathological range. In exemplary aspects, the subject is one who has anumber of polyQ tracts in the 47^(th) exon of the CACNA1A gene which isconsidered pathological (e.g., 19 or more polyQ tracts).

In exemplary aspects, the subject is a subject who has more than 10(e.g., more than 11, 12, 13, 14, 15, 16, 17, 18) polyQ tracts in the47^(th) exon of the CACNA1A gene. In exemplary aspects, the subject is asubject who has more than 15 (e.g., 16, 17, 18) polyQ tracts in the47^(th) exon of the CACNA1A gene.

In exemplary aspects, the method of treating a subject with SCA6 or apredisposition to SCA6 comprises administering to the subject (i) anantisense molecule that binds to a portion of an IRES of a CACNA1A genecomprising the sequence of SEQ ID NO: 180, optionally, wherein theantisense molecule binds to Argonaute 4 (Ago4), (ii) a vector encodingthe antisense molecule, (iii) a cell comprising the vector or antisensemolecule, (iv) a extracellular vesicle comprising the antisensemolecule, or (v) a combination thereof. Optionally, the vector is arecombinant expression vector of as described herein, the cell is a cellas described herein, or the extracellular vesicle is an extracellularvesicle as described herein. In exemplary aspects, the antisensemolecule comprises the sequence of SEQ ID NO: 179. In exemplary aspects,the vector is a recombinant adeno-associated viral (AAV) vector, e.g.,an AAV serotype 9 (AAV9) vector. In exemplary aspects, the recombinantAAV vector comprises one or more of a promoter, a pair of invertedterminal repeats (ITRs), and a polyadenylation signal sequence. Inexemplary aspects, the promoter is a human cytomegalovirus (CMV)immediate early promoter. In exemplary aspects, the ITRs are AAV ITRs.In exemplary aspects, the polyadenylation signal sequence is an simianvirus 40 (SV40) polyadenylation signal sequence. In exemplary aspects,the recombinant expression vector comprises a woodchuck hepatitis virusposttranscriptional regulatory element (WPRE). In exemplary aspects, therecombinant expression vector comprises an ITR upstream of the human CMVimmediate early promoter, which is located upstream of the nucleotidesequence encoding the antisense molecule, which is located upstream ofthe WPRE which is located upstream of the SV40 polyadenylation signalsequence which is located upstream of the the other ITR.

The term “treat” as well as words stemming therefrom, as used herein, donot necessarily imply 100% or complete treatment. Rather, there arevarying degrees of treatment of which one of ordinary skill in the artrecognizes as having a potential benefit or therapeutic effect. In thisrespect, the inventive methods can provide any amount, level, or degreeof treatment in a subject. Furthermore, the treatment provided by themethods of the invention may include treatment of one or more conditionsor symptoms or signs of the genetic disease, trinucleotide repeatdisorder, polyQ disease, or SCA6 being treated. For example, the methodof the invention may address one or more of: episodes of ataxia, vertigoand/or osscilopsia, diplopia, REM sleep disorders, dysarthria,dysphagia, ambulation and mobility difficulties, sleep apnea, ataxia,speech difficulties, involuntary eye movements (nystagmus), doublevision, loss of coordination in their arms, tremors, uncontrolled muscletensing (dystonia), severe incapacitation, aspiration pneumonia orrespiratory failure. Also, the treatment provided by the methods of theinvention may encompass slowing the progression of the disease ordisorder. For example, the treatment may slow the progression of one ormore of: episodes of ataxia, vertigo and/or osscilopsia, diplopia, REMsleep disorders, dysarthria, dysphagia, ambulation and mobilitydifficulties, sleep apnea, ataxia, speech difficulties, involuntary eyemovements (nystagmus), double vision, loss of coordination in theirarms, tremors, uncontrolled muscle tensing (dystonia), severeincapacitation, aspiration pneumonia or respiratory failure.

Accordingly, the invention provides methods of inhibiting or preventingPurkinje cell degeneration in a subject. The invention also accordinglyprovides methods of inhibiting or preventing ataxia in a subject. Inexemplary embodiments, the methods comprise administering to the subject(i) an antisense molecule that binds to a portion of an IRES of aCACNA1A gene comprising the sequence of SEQ ID NO: 180, optionally,wherein the antisense molecule binds to Argonaute 4 (Ago4), (ii) avector encoding the antisense molecule, (iii) a cell comprising thevector or antisense molecule, (iv) a extracellular vesicle comprisingthe antisense molecule, or (v) a combination thereof. Optionally, thevector is a recombinant expression vector of as described herein, thecell is a cell as described herein, or the extracellular vesicle is anextracellular vesicle as described herein. In exemplary aspects, theantisense molecule comprises the sequence of SEQ ID NO: 179. Inexemplary aspects, the vector is a recombinant adeno-associated viral(AAV) vector, e.g., an AAV serotype 9 (AAV9) vector. In exemplaryaspects, the recombinant AAV vector comprises one or more of a promoter,a pair of inverted terminal repeats (ITRs), and a polyadenylation signalsequence. In exemplary aspects, the promoter is a human cytomegalovirus(CMV) immediate early promoter. In exemplary aspects, the ITRs are AAVITRs. In exemplary aspects, the polyadenylation signal sequence is ansimian virus 40 (SV40) polyadenylation signal sequence. In exemplaryaspects, the recombinant expression vector comprises a woodchuckhepatitis virus posttranscriptional regulatory element (WPRE). Inexemplary aspects, the recombinant expression vector comprises an ITRupstream of the human CMV immediate early promoter, which is locatedupstream of the nucleotide sequence encoding the antisense molecule,which is located upstream of the WPRE which is located upstream of theSV40 polyadenylation signal sequence which is located upstream of thethe other ITR.

Polyglutamine (PolyQ) Diseases

Because SCA6 is a polyQ disease, the steps of the methods of treatingSCA6 provided herein are proposed as being useful in the treatment ofother polyQ diseases. Accordingly, the invention provides methods oftreating a polyglutamine (PolyQ) disease in a subject in need thereof.In exemplary embodiments, the methods comprise the step of administeringto the subject an IRES inhibitor in an amount effective for treating thepolyQ disease in the subject. In exemplary embodiments, the methodscomprise administering to the subject (i) an antisense molecule thatbinds to a portion of an IRES of a CACNA1A gene comprising the sequenceof SEQ ID NO: 180, optionally, wherein the antisense molecule binds toArgonaute 4 (Ago4), (ii) a vector encoding the antisense molecule, (iii)a cell comprising the vector or antisense molecule, (iv) a extracellularvesicle comprising the antisense molecule, or (v) a combination thereof.Optionally, the vector is a recombinant expression vector of asdescribed herein, the cell is a cell as described herein, or theextracellular vesicle is an extracellular vesicle as described herein.In exemplary aspects, the antisense molecule comprises the sequence ofSEQ ID NO: 179. In exemplary aspects, the vector is a recombinantadeno-associated viral (AAV) vector, e.g., an AAV serotype 9 (AAV9)vector. In exemplary aspects, the recombinant AAV vector comprises oneor more of a promoter, a pair of inverted terminal repeats (ITRs), and apolyadenylation signal sequence. In exemplary aspects, the promoter is ahuman cytomegalovirus (CMV) immediate early promoter. In exemplaryaspects, the ITRs are AAV ITRs. In exemplary aspects, thepolyadenylation signal sequence is an simian virus 40 (SV40)polyadenylation signal sequence. In exemplary aspects, the recombinantexpression vector comprises a woodchuck hepatitis virusposttranscriptional regulatory element (WPRE). In exemplary aspects, therecombinant expression vector comprises an ITR upstream of the human CMVimmediate early promoter, which is located upstream of the nucleotidesequence encoding the antisense molecule, which is located upstream ofthe WPRE which is located upstream of the SV40 polyadenylation signalsequence which is located upstream of the the other ITR.

As used herein, the term “polyQ disease” refers to a trinucleotiderepeat disorder in which the codon CAG is repeated in the coding regionof a gene resulting in a polyQ tract beyond a normal or standard. PolyQdiseases known to date include those listed in the table below.

No. of PolyQ No. of PolyQ repeats in repeats in PolyQ Disease GeneNormal State Pathogenic State DRPLA ATN1 or  6-35 49-88 (Dentatorubro-DRPLA pallidoluysian atrophy) HD (Huntington's HTT 10-35 35+ disease)(Huntingtin) SBMA Androgen  9-36 38-62 (Spinobulbar receptor on muscularatrophy or the X Kennedy disease) chromosome. SCA1 ATXN1  6-35 49-88(Spinocerebellar ataxia Type 1) SCA2 ATXN2 14-32 33-77 (Spinocerebellarataxia Type 2) SCA3 (Spinocerebellar ATXN3 12-40 55-86 ataxia Type 3 orMachado-Joseph disease) SCA6 CACNA1A  4-18 21-30 (Spinocerebellar ataxiaType 6) SCA7 ATXN7  7-17 38-120 (Spinocerebellar ataxia Type 7) SCA17(Spinocerebellar TBP 25-42 47-63 ataxia Type 17) Source: “Trinucleotiderepeat disorder” on Wickipedia, 2013

Besides being encoded by a gene comprising repeated CAG codons, thepolyQ proteins involved in polyQ diseases have no structural similarityotherwise. Several polyQ proteins have nuclear functions relating togene regulation (Riley B E, et al., Genes Dev. 2006; 20(16):2183-92).Toxicity in these disorders depends on the flanking protein context ofthe polyQ tract, and is frequently associated with transport to thenucleus of the full length or a toxic fragment of the mutant protein (LaSpada A R, et al. Curr Med Chem. 2010; 17(27):3058-68, Robertson et al.,Curr Med Chem (2010) 17(27): 3058-3068).

Neurodegenerative Diseases (ND)

Neurodegenerative diseases are defined as hereditary and sporadicconditions which are characterized by progressive nervous systemdysfunction. These disorders are often associated with atrophy of theaffected central or peripheral structures of the nervous system. Theyinclude diseases such as Alzheimer's Disease and other dementias, BrainCancer, Degenerative Nerve Diseases, Encephalitis, Epilepsy, GeneticBrain Disorders, Head and Brain Malformations, Hydrocephalus, Stroke,Parkinson's Disease, Multiple Sclerosis, Amyotrophic Lateral Sclerosis(ALS or Lou Gehrig's Disease), Huntington's Disease, Prion Diseases, andothers.

There are extensive overlaps between SCA and other neurodegenerativediseases (NDs) (Shan J, Diamond M I. Hum Mol Genet. 2007; 16 Spec No.2:R115-23; Bauer P O, Nukina N. J Neurochem. 2009; 110(6):1737-65; LaSpada A R, Taylor J P. Nat Rev Genet. 2010; 11(4):247-58; He X H, Lin F,Qin Z H. Neurosci Bull. 2010; 26(3):247-56). Although neuronal cell lossis most evident in the regions responsible for the principal clinicalpresentation of NDs, neurodegeneration is nearly always more widespread(Gomez C M, et al. Ann Neurol. 1997; 42(6):933-50; Koeppen A H. Journalof Neuropathology & Experimental Neurology. 1998; 57(6):531-43; Havel LS et al. Mol Brain. 2009; 2:21; Pula J H, et al., 2011; 35(3):108-14).Moreover, there is an increasing overlap in possible disease mechanisms.For example, there are growing mechanistic genetic overlaps between ALSand SCA2 (24) and SCA6 and epilepsy (Yalcin O. Seizure. 2011,Rajakulendran S, et al., 2012). Thus, insights into molecularpathogenesis in each disease will have a wider impact on understandingneuronal death and dysfunction in other systems.

The steps of the method of treating SCA6 provided herein are proposed asbeing useful in the treatment of other neurodegenerative diseases.Accordingly, the invention provides methods of treating aneurodegenerative disease. In exemplary aspects, the method comprisesthe step of administering to the subject an IRES inhibitor in an amounteffective for treating the neurodegenerative disease. In exemplaryaspects, the neurodegenerative disease is episodic ataxia type 2,familial hemiplegic migraine, and epilepsy, Alzheimer's Disease andother dementias, Brain Cancer, Degenerative Nerve Diseases,Encephalitis, Epilepsy, Genetic Brain Disorders, Head and BrainMalformations, Hydrocephalus, Stroke, Parkinson's Disease, MultipleSclerosis, Amyotrophic Lateral Sclerosis (ALS or Lou Gehrig's Disease),Huntington's Disease, Prion Diseases, and others. In exemplary aspects,the neurodegenerative disease is SCA6.

In exemplary embodiments, the methods comprise administering to thesubject (i) an antisense molecule that binds to a portion of an IRES ofa CACNA1A gene comprising the sequence of SEQ ID NO: 180, optionally,wherein the antisense molecule binds to Argonaute 4 (Ago4), (ii) avector encoding the antisense molecule, (iii) a cell comprising thevector or antisense molecule, (iv) a extracellular vesicle comprisingthe antisense molecule, or (v) a combination thereof. Optionally, thevector is a recombinant expression vector of as described herein, thecell is a cell as described herein, or the extracellular vesicle is anextracellular vesicle as described herein. In exemplary aspects, theantisense molecule comprises the sequence of SEQ ID NO: 179. Inexemplary aspects, the vector is a recombinant adeno-associated viral(AAV) vector, e.g., an AAV serotype 9 (AAV9) vector. In exemplaryaspects, the recombinant AAV vector comprises one or more of a promoter,a pair of inverted terminal repeats (ITRs), and a polyadenylation signalsequence. In exemplary aspects, the promoter is a human cytomegalovirus(CMV) immediate early promoter. In exemplary aspects, the ITRs are AAVITRs. In exemplary aspects, the polyadenylation signal sequence is ansimian virus 40 (SV40) polyadenylation signal sequence. In exemplaryaspects, the recombinant expression vector comprises a woodchuckhepatitis virus posttranscriptional regulatory element (WPRE). Inexemplary aspects, the recombinant expression vector comprises an ITRupstream of the human CMV immediate early promoter, which is locatedupstream of the nucleotide sequence encoding the antisense molecule,which is located upstream of the WPRE which is located upstream of theSV40 polyadenylation signal sequence which is located upstream of thethe other ITR.

IRES Inhibitors

The methods of the invention comprise the step of administering to asubject an IRES inhibitor. As used herein, the term “IRES” is synonymouswith “internal ribosome entry site” and refers to a nucleotide sequencethat allows for the initiation of translation in the middle of amessenger ribonucleic acid (mRNA) sequence as part of the greaterprocess of protein synthesis. IRES-mediated translation in exemplaryembodiments is considered as “cap-independent” translation. In exemplaryaspects, the IRES is located in the 5′ untranslated region (5′ UTR) of agene. In alternative aspects, the IRES is located within the middle of amRNA.

As used herein, the term “IRES inhibitor” refers to any compound thatinhibits IRES-mediated activity, e.g., IRES-mediated translation of anmRNA or IRES protein binding. In exemplary embodiments, the IRESinhibitor is a nucleic acid, a nucleic acid analog, a peptide, apolypeptide, a peptidomimetic, a peptoid, a small molecular weightcompound, or the like. In exemplary aspects, the IRES inhibitor is anantisense molecule that binds to mRNA produced by a gene which gene isknown to be causative of a particular disease. In exemplary aspects, theantisense molecule is an antisense oligonucleotide comprisingdeoxyribonucleic acid (DNA) and/or ribonucleic acid (RNA). In exemplaryaspects, the antisense molecule is an antisense nucleic acid analogcomprising a structural analog of DNA and/or RNA. In exemplary aspects,the antisense molecule is synthetic. As used herein, the term“synthetic” refers to a manufactured or engineered compound or moleculewhich does not occur or exist in nature. In alternative exemplaryaspects, the IRES inhibitor is a small molecular weight compound havinga molecular weight of less than about 10 kDa, as measured by, forexample, gel filtration chromatography. One skilled in the art willappreciate that a small molecular weight compound can be a non-peptidiccompound that is cell-permeable and resistant to degradation. The term“non-peptidic” as used herein refers to not being derived from aprotein. The IRES inhibitor may be natural, synthetic, or semi-syntheticor partially synthetic.

The IRES inhibitor may inhibit any one or more of IRES-mediatedactivities. In exemplary aspects, the IRES inhibitor inhibitsIRES-mediated translation of an mRNA and/or IRES-mediated proteinbinding. For example, the IRES inhibitor may be a compound which blocksthe binding of an IRES trans-acting factor (ITAF) to an IRES. Inexemplary aspects, the IRES inhibitor binds to or adjacent to the IRESand effectively blocks the binding of the ITAF to the IRES. Inalternative aspects, the IRES inhibitor binds to the ITAF and blocks thebinding of the ITAF to the IRES. The IRES inhibitor may binds to theITAF within or adjacent to the IRES-binding site of the ITAF, therebyblocking the ITAF's ability to bind to the IRES.

In additional or alternative embodiments, the IRES inhibitor blockstranslation of an mRNA through additional or alternative mechanisms. Inexemplary aspects, the IRES inhibitor alters the primary, secondary,and/or tertiary structure of the IRES. In exemplary aspects, the IRESinhibitor effects the change in structure of the IRES by cleaving withinthe IRES or modifying the chemico-physico attributes of the IRES.

The IRES inhibitor may provide any level of inhibition of IRES-mediatedactivity. In exemplary aspects, the IRES inhibitor inhibits at least 10%IRES-mediated activity. In exemplary aspects, the IRES inhibitorachieves at least a 50% inhibition of IRES-mediated activity. Inexemplary aspects, the IRES inhibitor achieves at least a 90% inhibitionof IRES-mediated activity. In exemplary aspects, the IRES inhibitorinhibits at least 10% IRES-mediated translation. In exemplary aspects,the IRES inhibitor achieves at least a 50% inhibition of IRES-mediatedtranslation. In exemplary aspects, the IRES inhibitor achieves at leasta 90% inhibition of IRES-mediated translation. Methods of testingIRES-mediated translation, and thus, methods of inhibiting IRES-mediatedtranslation, are known in the art. See, for example, Du et al., Cell154: 118-133 (2013) and Examples 1 and 2 herein. In exemplary aspects,methods of testing the inhibition of IRES-mediated translation comprisesuse of one or more bi-cistronic reporter constructs. In exemplaryaspects, the IRES inhibitor inhibits at least 10% IRES-mediated proteinbinding, e.g., IRES-protein binding. In exemplary aspects, the IRESinhibitor achieves at least a 50% inhibition of IRES-protein binding. Inexemplary aspects, the IRES inhibitor achieves at least a 90% inhibitionof IRES-protein binding. Methods of testing levels of IRES-proteinbinding are known in the art and include for example electrophoreticmobility shift assay (EMSA). See, e.g., Ausubel, Frederick M. (1994).Current Protocols in molecular biology. Chichester: John Wiley & Sons.pp. 12.2.1-11, and Example 1 herein.

In exemplary aspects, the IRES inhibitor targets an IRES within a viralgenome. In exemplary aspects, the IRES inhibitor does not target an IRESwithin a viral genome. IRESs found within a viral genome are known inthe art and include, for example, a picornavirus IRES, aphthovirus IRES,Hepatitis A IRES, Hepatitis C IRES, Pestivirus IRES, Cripavirus IRES,Kaposi's sarcoma associated herpesvirus IRES, and the Marek's diseasevirus IRES. The IRES inhibitor in exemplary aspects targets an IRESwithin a poliovirus genome, a rhinovirus genome, an encephalomyocarditisvirus genome, a foot-and-mouth disease virus genome, a Hepatitis A virusgenome, a Hepatitis C virus genome, a classical swine fever virusgenome, a bovine viral diarrhea virus genome, a friend murine leukemiavirus genome, a Moloney murine leukemia virus genome, a Rous sarcomavirus genome, a Human immunodeficiency virus genome, a Plautia staliintestine virus genome, a Rhopalosiphum padi virus genome, a Cricketparalysis virus genome, a Triatoma virus genome, a Kaposi's sarcomaassociated herpes virus genome, or a Marek's disease virus genome.

In exemplary aspects, the IRES inhibitor targets an IRES within acellular mRNA. In exemplary aspects, the IRES inhibitor does not targetan IRES within a cellular mRNA. In exemplary aspects, the cellular mRNAencodes a growth factor, a transcription factor, a translation factor,an oncogene, a transporter, a receptor, an activator of apoptosis, or aprotein localized in neuronal dendrites. In exemplary aspects, the IRESinhibitor targets or does not target an mRNA encoding any one or more ofthe following proteins: Fibroblast growth factor (FGF-1, FGF-2),Platelet-derived growth factor B (PDGF/c-sis), Vascular endothelialgrowth factor (VEGF), Insulin-like growth factor 2 (IGF-II), theAntennapedia, Ultrabithorax, MYT-2, NF-κB repressing factor NRF,AML1/RUNX1, Gtx homeodomain protein, Eukaryotic initiation factor 4G(e1F4G)a, Eukaryotic initiation factor 4G1 (e1F4G1)a, Death associatedprotein 5 (DAPS), c-myc, L-myc, Pim-1, Protein kinase p58PITSLRE, p53,Cationic amino acid transporter Cat-1, Nuclear form of Notch 2,Voltage-gated potassium channel, Apoptotic protease activating factor(Apaf-1), X-linked inhibitor of apoptosis (XIAP), HIAP2, Bc1-xL, Bcl-2,Activity-regulated cytoskeletal protein (ARC), a-subunit of calciumcalmodulin dependent kinase II dendrin, Microtubule-associated protein 2(MAP2), neurogranin (RC3), Amyloid precursor protein, Immunoglobulinheavy chain binding protein (BiP), Heat shock protein 70, β-subunit ofmitochondrial H+-ATP synthase, Ornithine decarboxylase, connexins 32 and43, HIF-1α, APC.

In exemplary aspects, the IRES inhibitor targets the IRES of a geneassociated with a diseased state. In exemplary aspects, the IRESinhibitor targets the IRES of a gene associated with a genetic disorder,e.g., a trinucleotide repeat disorder. In exemplary aspects, the IRESinhibitor targets the IRES of a gene associated with a polyglutaminedisease. Genes associated with a polyQ disease are described herein.See, e.g., the table in the section entitled “Polyglutamine Diseases.”

In exemplary aspects, the IRES inhibitor targets the IRES of an mRNAencoding a calcium channel or a transcription factor. In exemplaryaspects, the IRES inhibitor targets the IRES of a bi-cistronic mRNAencoding both a calcium channel and a transcription factor. In exemplaryaspects, the IRES inhibitor targets the IRES of the α1A mRNA, which isencoded by the CACNA1A gene. The CACNA1A gene, officially named as thecalcium channel, voltage-dependent, P/Q type, alpha 1A subunit gene, isdescribed in the Gene database of the National Center for BiotechnologyInformation (NCBI) as Gene ID 773. The gene encodes 5 isoforms of theα1A protein, a voltage-gated calcium channel subunit. Each are providedin the GenBank database as follows: α1A Isoform 1 (NP_000059); α1AIsoform 2 (NP_075461.2),α1A Isoform 3 (NP_001120693.1), α1A Isoform 4(NP_001120694.1), and α1A Isoform 5 (NP_001167551.1). While the IRES ispresent in all five isoforms, the mRNA encoding Isoforms 1, 3, and 5 donot have the polyQ tract, whereas the mRNA encoding Isoforms 2 and 4comprise the polyQ tract. In exemplary aspects, the IRES inhibitortargets the IRES of the mRNA of the α1A Isoform 2 or of the α1A Isoform4. The CACNA1A gene also encodes the α1ACT protein, a transcriptionfactor that coordinates expression of a program of genes involved inneural and Purkinje cell development. The sequence of the nucleic acidencoding the α1ACT protein is known in the art and is set forth hereinas SEQ ID NO: 4.

In exemplary aspects, the IRES inhibitor blocks binding of the IRES ofthe α1A mRNA to an IRES ITAF which binds to the IRES of the α1A mRNA. Inexemplary aspects, the IRES inhibitor binds to an ITAF that binds to theIRES of the α1A mRNA. In exemplary aspects, the IRES inhibitor binds toor adjacent to the IRES of the α1A mRNA.

In exemplary aspects, the IRES inhibitor is an antisense molecule whichpermits specific suppression or reduction of expression of the nucleicacid (e.g., the mRNA) encoding the α1ACT protein. In exemplary aspects,the IRES inhibitor is an antisense molecule which permits specificsuppression or reduction of expression of the nucleic acid (e.g., themRNA) encoding the α1ACT protein without affecting the expression of thenucleic acid of the α1A protein. In exemplary aspects, the IRESinhibitor causes specific suppression of translation of the nucleic acid(e.g., the mRNA) encoding the α1ACT protein without causing degradationof the α1A mRNA and/or without inhibition of expression of the α1Aprotein.

In exemplary aspects, the antisense molecule can be complementary to theentire coding region of the nucleic acid encoding the α1ACT protein (SEQID NO: 4), or to a portion thereof. The antisense molecule in exemplaryaspects is about 5, about 10, about 15, about 20, about 25, about 30,about 35, about 40, about 45 or about 50 nucleotides in length.

In exemplary aspects, the antisense molecule is about X to about Ynucleotides in length, wherein X is 10, 11, 12, 13, 14, or 15 and Y is20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. In exemplary aspects, theantisense molecule is about 10 to about 20 nucleotides in length, about10 to about 21 nucleotides in length, about 10 to about 22 nucleotidesin length, about 10 to about 23 nucleotides in length, about 10 to about24 nucleotides in length, about 10 to about 25 nucleotides in length,about 10 to about 26 nucleotides in length, about 10 to about 27nucleotides in length, about 10 to about 28 nucleotides in length, about10 to about 29 nucleotides in length, or about 10 to about 30nucleotides in length. In exemplary aspects, the antisense molecule isabout 11 to about 20 nucleotides in length, about 11 to about 21nucleotides in length, about 11 to about 22 nucleotides in length, about11 to about 23 nucleotides in length, about 11 to about 24 nucleotidesin length, about 11 to about 25 nucleotides in length, about 11 to about26 nucleotides in length, about 11 to about 27 nucleotides in length,about 11 to about 28 nucleotides in length, about 11 to about 29nucleotides in length, or about 11 to about 30 nucleotides in length. Inexemplary aspects, the antisense molecule is about 12 to about 20nucleotides in length, about 12 to about 21 nucleotides in length, about12 to about 22 nucleotides in length, about 12 to about 23 nucleotidesin length, about 12 to about 24 nucleotides in length, about 12 to about25 nucleotides in length, about 12 to about 26 nucleotides in length,about 12 to about 27 nucleotides in length, about 12 to about 28nucleotides in length, about 12 to about 29 nucleotides in length, orabout 12 to about 30 nucleotides in length. In exemplary aspects, theantisense molecule is about 13 to about 20 nucleotides in length, about13 to about 21 nucleotides in length, about 13 to about 22 nucleotidesin length, about 13 to about 23 nucleotides in length, about 13 to about24 nucleotides in length, about 13 to about 25 nucleotides in length,about 13 to about 26 nucleotides in length, about 13 to about 27nucleotides in length, about 13 to about 28 nucleotides in length, about13 to about 29 nucleotides in length, or about 13 to about 30nucleotides in length. In exemplary aspects, the antisense molecule isabout 14 to about 20 nucleotides in length, about 14 to about 21nucleotides in length, about 14 to about 22 nucleotides in length, about14 to about 23 nucleotides in length, about 14 to about 24 nucleotidesin length, about 14 to about 25 nucleotides in length, about 14 to about26 nucleotides in length, about 14 to about 27 nucleotides in length,about 14 to about 28 nucleotides in length, about 14 to about 29nucleotides in length, or about 14 to about 30 nucleotides in length. Inexemplary aspects, the antisense molecule is about 15 to about 20nucleotides in length, about 15 to about 21 nucleotides in length, about15 to about 22 nucleotides in length, about 15 to about 23 nucleotidesin length, about 15 to about 24 nucleotides in length, about 15 to about25 nucleotides in length, about 15 to about 26 nucleotides in length,about 15 to about 27 nucleotides in length, about 15 to about 28nucleotides in length, about 15 to about 29 nucleotides in length, orabout 15 to about 30 nucleotides in length. In exemplary aspects, theantisense molecule is about 15 to about 30 nucleotides in length orabout 20 to 30 nucleotides in length or about 25 to 30 nucleotides inlength. In exemplary aspects, the antisense molecule is about 25nucleotides in length.

In exemplary aspects, the antisense molecule binds to at least a portionof the sequence of SEQ ID NO: 5 of the α1A mRNA. SEQ ID NO: 5 is aportion of the sequence of the nucleic acid encoding the α1ACT protein(known in the art as GenBank Accession No. NM_01127222 and providedherein as SEQ ID NO: 4. SEQ ID NO: 5 is 5101 through 6110 by of GenBankAccession No. NM_01127222 (SEQ ID NO: 4)). In exemplary aspects, theantisense molecule binds to at least a portion of the sequence of SEQ IDNO: 5 of the α1A mRNA, wherein the portion is at least 5 contiguousnucleotides (or at least 2 or at least 3 contiguous nucleotides) of SEQID NO: 5. In exemplary aspects, the antisense molecule binds to at least15 contiguous nucleotides of the sequence of SEQ ID NO: 5. In exemplaryaspects, the antisense molecule binds to at least 20 contiguousnucleotides of the sequence of SEQ ID NO: 5. In exemplary aspects, theantisense molecule binds to at least 25 contiguous nucleotides of thesequence of SEQ ID NO: 5. In exemplary aspects, the antisense moleculebinds to a portion of the α1A mRNA (and for purposes herein, thisportion is termed hereinafter as a target sequence) and the targetsequence comprises the ATG start site (i.e., start codon) of thesequence encoding α1ACT (plus flanking sequence upstream and/ordownstream of the ATG start site). The coding sequence of α1ACT isprovided herein as SEQ ID NO: 12 and the α1A mRNA comprising the codingsequence and the ATG start site is provided herein as SEQ ID NO: 4. Inexemplary aspects, the antisense molecule binds to a target sequence ofthe α1A mRNA, which target sequence is located upstream or 5′ to the ATGstart codon of the sequence encoding α1ACT. In exemplary aspects, theantisense molecule binds to a target sequence which is located withinthe 200 nucleotides immediately upstream or 5′ to the ATG start codon ofthe sequence encoding α1ACT. In exemplary aspects, the antisensemolecule binds to a target sequence which is located within the 100nucleotides immediately upstream or 5′ to the ATG start codon of thesequence encoding α1ACT. In exemplary aspects, the antisense moleculebinds to a target sequence which is located within the 50 nucleotidesimmediately upstream or 5′ to the ATG start codon of the sequenceencoding α1ACT. In exemplary aspects, the antisense molecule binds to atarget sequence which is located within the 25 nucleotides immediatelyupstream or 5′ to the ATG start codon of the sequence encoding α1ACT. Inexemplary aspects, the antisense molecule binds to a target sequencewhich is located within the 15 nucleotides immediately upstream or 5′ tothe ATG start codon of the sequence encoding α1ACT. In exemplaryaspects, the antisense molecule binds to a target sequence of the α1AmRNA, which target sequence is located downstream or 3′ to the ATG startcodon of the sequence encoding α1ACT. In exemplary aspects, theantisense molecule binds to a target sequence which is located withinthe 200 nucleotides immediately downstream or 3′ to the ATG start codonof the sequence encoding α1ACT. In exemplary aspects, the antisensemolecule binds to a target sequence which is located within the 100nucleotides immediately downstream or 3′ to the ATG start codon of thesequence encoding α1ACT. In exemplary aspects, the antisense moleculebinds to a target sequence which is located within the 50 nucleotidesimmediately downstream or 3′ to the ATG start codon of the sequenceencoding α1ACT. In exemplary aspects, the antisense molecule binds to atarget sequence which is located within the 25 nucleotides immediatelydownstream or 3′ to the ATG start codon of the sequence encoding α1ACT.In exemplary aspects, the antisense molecule binds to a target sequencewhich is located within the 15 nucleotides immediately downstream or 3′to the ATG start codon of the sequence encoding α1ACT.

In exemplary aspects, the antisense molecule binds to at least a portionof the sequence of SEQ ID NO: 6 of the α1A mRNA. In exemplary aspects,the antisense molecule binds to at least a portion of the sequence ofSEQ ID NO: 6 of the α1A mRNA, wherein the portion is at least 3contiguous nucleotides (or at least 5 or at least 2 contiguousnucleotides) of SEQ ID NO: 6. In exemplary aspects, the antisensemolecule binds to at least 15 contiguous nucleotides of the sequence ofSEQ ID NO: 6. In exemplary aspects, the antisense molecule binds to atleast 20 contiguous nucleotides of the sequence of SEQ ID NO: 6. Inexemplary aspects, the antisense molecule binds to at least 25contiguous nucleotides of the sequence of SEQ ID NO: 6. In exemplaryaspects, the antisense molecule binds to at least a portion of thesequence of SEQ ID NO: 7 of the α1A mRNA. In exemplary aspects, theantisense molecule binds to at least a portion of the sequence of SEQ IDNO: 7 of the α1A mRNA, wherein the portion is at least 2 contiguousnucleotides (or at least 5 or at least 3 contiguous nucleotides) of SEQID NO: 7. In exemplary aspects, the antisense molecule binds to theentire sequence of SEQ ID NO: 7.

In exemplary aspects, the antisense molecule is an antisenseoligonucleotide or antisense nucleic acid analog which is complementaryto at least a portion of the sequence of SEQ ID NO: 5, SEQ ID NO: 6, orSEQ ID NO: 7. The antisense molecule in some aspects is complementary toat least 15 contiguous bases of said sequence. The antisense molecule insome aspects is complementary to at least 20 contiguous bases of thesequence of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. The antisensemolecule in some aspects is complementary to at least 25 contiguousbases of the sequence of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. Inexemplary aspects, the antisense molecule is an antisenseoligonucleotide or antisense nucleic acid analog comprising at least 15contiguous bases of SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11, whichare complementary sequences of SEQ ID NOs: 5-7, respectively. Inexemplary aspects, the antisense molecule is an antisenseoligonucleotide or antisense nucleic acid analog comprising at least 15contiguous bases that differs by not more than 3 bases from a portion of15 contiguous bases of SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11. Inexemplary aspects, the antisense molecule is an antisenseoligonucleotide or antisense nucleic acid analog comprising at least 15contiguous bases that is at least 90% identical to a portion of 15contiguous bases of SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11. Inexemplary aspects, the antisense molecule is an antisenseoligonucleotide or antisense nucleic acid analog comprising, consistingessentially of, or consisting of SEQ ID NO: 8.

In exemplary aspects, the antisense molecule binds to at least a portionof the sequence of SEQ ID NO: 56 of the α1A mRNA. In exemplary aspects,the antisense molecule binds to at least a portion of the sequence ofSEQ ID NO: 56 of the α1A mRNA, wherein the portion is at least 2contiguous nucleotides (or at least 5 or at least 3 contiguousnucleotides) of SEQ ID NO: 56. In exemplary aspects, the antisensemolecule binds to the entire sequence of SEQ ID NO: 56. In exemplaryaspects, the antisense molecule binds to at least a portion of thesequence of SEQ ID NO: 57 of the α1A mRNA. In exemplary aspects, theantisense molecule binds to at least a portion of the sequence of SEQ IDNO: 57 of the α1A mRNA, wherein the portion is at least 2 contiguousnucleotides (or at least 5 or at least 3 contiguous nucleotides) of SEQID NO: 57. In exemplary aspects, the antisense molecule binds to theentire sequence of SEQ ID NO: 57.

In exemplary aspects, the antisense molecule is an antisenseoligonucleotide or antisense nucleic acid analog which is complementaryto at least a portion of the sequence of SEQ ID NO: 56 or SEQ ID NO: 57.The antisense molecule in some aspects is complementary to at least 15contiguous bases of said sequence. The antisense molecule in someaspects is complementary to at least 20 contiguous bases of the sequenceof SEQ ID NO: 56 or SEQ ID NO: 57. The antisense molecule in someaspects is complementary to at least 25 contiguous bases of the sequenceof SEQ ID NO: 56. In exemplary aspects, the antisense molecule is anantisense oligonucleotide or antisense nucleic acid analog comprising atleast 15 contiguous bases of SEQ ID NO: 58 or SEQ ID NO: 59, which arecomplementary sequences of SEQ ID NOs: 56 and 57, respectively. Inexemplary aspects, the antisense molecule is an antisenseoligonucleotide or antisense nucleic acid analog comprising at least 15contiguous bases that differs by not more than 3 bases from a portion of15 contiguous bases of SEQ ID NO: 58 or SEQ ID NO: 59. In exemplaryaspects, the antisense molecule is an antisense oligonucleotide orantisense nucleic acid analog comprising at least 15 contiguous basesthat is at least 90% identical to a portion of 15 contiguous bases ofSEQ ID NO: 58 or SEQ ID NO: 59. In exemplary aspects, the antisensemolecule is an antisense oligonucleotide or antisense nucleic acidanalog comprising, consisting essentially of, or consisting of SEQ IDNO: 55.

In exemplary aspects, the antisense molecule binds to at least a portionof the sequence of SEQ ID NO: 180. In exemplary aspects, the antisensemolecule binds to at least a portion of the sequence of SEQ ID NO: 180of the α1A mRNA, wherein the portion is at least 5 contiguousnucleotides (or at least 2 or at least 3 contiguous nucleotides) of SEQID NO: 180. In exemplary aspects, the antisense molecule binds to atleast 15 contiguous nucleotides of the sequence of SEQ ID NO: 180. Inexemplary aspects, the antisense molecule binds to at least 20contiguous nucleotides of the sequence of SEQ ID NO: 180. In exemplaryaspects, the antisense molecule binds to at least 25 contiguousnucleotides of the sequence of SEQ ID NO: 180.

In exemplary aspects, the antisense molecule is an antisenseoligonucleotide or antisense nucleic acid analog which is complementaryto at least a portion of the sequence of SEQ ID NO: 180. The antisensemolecule in some aspects is complementary to at least 15 contiguousbases of said sequence. The antisense molecule in some aspects iscomplementary to at least 20 contiguous bases of the sequence of SEQ IDNO: 180. The antisense molecule in some aspects is complementary to atleast 25 contiguous bases of the sequence of SEQ ID NO: 180. Inexemplary aspects, the antisense molecule is an antisenseoligonucleotide or antisense nucleic acid analog comprising at least 15contiguous bases of SEQ ID NO: 179. In exemplary aspects, the antisensemolecule is an antisense oligonucleotide or antisense nucleic acidanalog comprising at least 15 contiguous bases that differs by not morethan 3 bases from a portion of 15 contiguous bases of SEQ ID NO: 179. Inexemplary aspects, the antisense molecule is an antisenseoligonucleotide or antisense nucleic acid analog comprising at least 15contiguous bases that is at least 90% identical to a portion of 15contiguous bases of SEQ ID NO: 179. In exemplary aspects, the antisensemolecule is an antisense oligonucleotide or antisense nucleic acidanalog comprising, consisting essentially of, or consisting of SEQ IDNO: 179.

The antisense molecule can be one which mediates RNA interference(RNAi). As known by one of ordinary skill in the art, RNAi is aubiquitous mechanism of gene regulation in plants and animals in whichtarget mRNAs are degraded in a sequence-specific manner (Sharp, GenesDev., 15, 485-490 (2001); Hutvagner et al., Curr. Opin. Genet. Dev., 12,225-232 (2002); Fire et al., Nature, 391, 806-811 (1998); Zamore et al.,Cell, 101, 25-33 (2000)). The natural RNA degradation process isinitiated by the dsRNA-specific endonuclease Dicer, which promotescleavage of long dsRNA precursors into double-stranded fragments between21 and 25 nucleotides long, termed small interfering RNA (siRNA; alsoknown as short interfering RNA) (Zamore, et al., Cell. 101, 25-33(2000); Elbashir et al., Genes Dev., 15, 188-200 (2001); Hammond et al.,Nature, 404, 293-296 (2000); Bernstein et al., Nature, 409, 363-366(2001)). siRNAs are incorporated into a large protein complex thatrecognizes and cleaves target mRNAs (Nykanen et al., Cell, 107, 309-321(2001). It has been reported that introduction of dsRNA into mammaliancells does not result in efficient Dicer-mediated generation of siRNAand therefore does not induce RNAi (Caplen et al., Gene 252, 95-105(2000); Ui-Tei et al., FEBS Lett, 479, 79-82 (2000)). The requirementfor Dicer in maturation of siRNAs in cells can be bypassed byintroducing synthetic 21-nucleotide siRNA duplexes, which inhibitexpression of transfected and endogenous genes in a variety of mammaliancells (Elbashir et al., Nature, 411: 494-498 (2001)).

In this regard, the IRES inhibitor in some aspects mediates RNAi and insome aspects is a siRNA molecule specific for inhibiting the expressionof the nucleic acid (e.g., the mRNA) encoding the α1ACT protein. Theterm “siRNA” as used herein refers to an RNA (or RNA analog) comprisingfrom about 10 to about 50 nucleotides (or nucleotide analogs) which iscapable of directing or mediating RNAi. In exemplary embodiments, ansiRNA molecule comprises about 15 to about 30 nucleotides (or nucleotideanalogs) or about 20 to about 25 nucleotides (or nucleotide analogs),e.g., 21-23 nucleotides (or nucleotide analogs). The siRNA can be doubleor single stranded, preferably double-stranded.

In alternative aspects, the IRES inhibitor is alternatively a shorthairpin RNA (shRNA) molecule specific for inhibiting the expression ofthe nucleic acid (e.g., the mRNA) encoding the α1ACT protein. The term“shRNA” as used herein refers to a molecule of about 20 or more basepairs in which a single-standed RNA partially contains a palindromicbase sequence and forms a double-strand structure therein (i.e., ahairpin structure). An shRNA can be an siRNA (or siRNA analog) which isfolded into a hairpin structure. shRNAs typically comprise about 45 toabout 60 nucleotides, including the approximately 21 nucleotideantisense and sense portions of the hairpin, optional overhangs on thenon-loop side of about 2 to about 6 nucleotides long, and the loopportion that can be, e.g., about 3 to 10 nucleotides long. The shRNA canbe chemically synthesized. Alternatively, the shRNA can be produced bylinking sense and antisense strands of a DNA sequence in reversedirections and synthesizing RNA in vitro with T7 RNA polymerase usingthe DNA as a template.

Though not wishing to be bound by any theory or mechanism it is believedthat after shRNA is introduced into a cell, the shRNA is degraded into alength of about 20 bases or more (e.g., representatively 21, 22, 23bases), and causes RNAi, leading to an inhibitory effect. Thus, shRNAelicits RNAi and therefore can be used as an effective component of theinvention. shRNA may preferably have a 3′-protruding end. The length ofthe double-stranded portion is not particularly limited, but ispreferably about 10 or more nucleotides, and more preferably about 20 ormore nucleotides. Here, the 3′-protruding end may be preferably DNA,more preferably DNA of at least 2 nucleotides in length, and even morepreferably DNA of 2-4 nucleotides in length.

In exemplary aspects, the antisense molecule is a microRNA (miRNA). Asused herein the term “microRNA” refers to a small (e.g., 15-22nucleotides), non-coding RNA molecule which base pairs with mRNAmolecules to silence gene expression via translational repression ortarget degradation. microRNA and the therapeutic potential thereof aredescribed in the art. See, e.g., Mulligan, MicroRNA: Expression,Detection, and Therapeutic Strategies, Nova Science Publishers, Inc.,Hauppauge, N.Y., 2011; Bader and Lammers, “The Therapeutic Potential ofmicroRNAs” Innovations in Pharmaceutical Technology, pages 52-55 (March2011); and Zhang et al., J Control Release 172(3): 962-974 (2013). Inexemplary aspects, the miRNA is a mature miRNA strand.

In exemplary aspects, the miRNA targets a portion of the IRES of theCACNA1A gene wherein the portion comprises sequence of SEQ ID NO: 180.As understood by the ordinarily skilled artisan, miRNA does not requireperfect pairing to its target. Thus, in exemplary aspects, the miRNAbinds to at least a portion of the sequence of SEQ ID NO: 180. Inexemplary aspects, the miRNA is complementary to at least a portion ofthe sequence of SEQ ID NO: 180. The miRNA in some aspects iscomplementary to at least 15 contiguous bases of said sequence. Inexemplary aspects, the miRNA comprises at least 15 contiguous bases ofSEQ ID NO: 179. In exemplary aspects, the miRNA comprises at least 15contiguous bases that differ by not more than 3 bases from a portion of15 contiguous bases of SEQ ID NO: 179. In exemplary aspects, the miRNAcomprises at least 15 contiguous bases that is at least 90% identical toa portion of 15 contiguous bases of SEQ ID NO: 179. In exemplaryaspects, the miRNA comprises, consists essentially of, or consists ofSEQ ID NO: 179. In exemplary aspects, the miRNA is about 15, about 16,about 17, about 18, about 19, about 20, about 21, about 22, about 23,about 24 nt in length.

In exemplary aspects, the antisense molecule is a precursor of miRNAcomprising SEQ ID NO: 179. In exemplary aspects, the antisense moleculeis a primary miRNA precursor (pri-miRNA) comprising SEQ ID NO: 179 whichis optionally, capped, polyadenylated, and/or comprises double-strandedstem-loop structures. In exemplary aspects, the antisense molecule is apre-miRNA precursor comprising SEQ ID NO: 179, which is optionally about70 to about 100 nt long and/or comprises a hairpin structure.

In exemplary aspects, the miRNA comprises the sequence of SEQ ID NO:179. In exemplary aspects, the miRNA comprises at least 15 contiguousbases of a sequence that (i) differs from SEQ ID NO: 179 by not morethan 3 bases (e.g., not more than 4, 5, 6, 7, 8, 9, 10 bases), (ii) isat least 90% (e.g., at least 93%, at least 95%, at least 98%, at least99%) identical to the sequence set forth in SEQ ID NO: 179, or (iii) iscompletely complementary to at least a portion of the sequence of SEQ IDNO: 180.

In exemplary aspects, the antisense molecule is an antisenseoligonucleotide comprising DNA or RNA or both DNA and RNA. In exemplaryaspects, the antisense oligonucleotide comprises naturally-occurringnucleotides and/or naturally-occurring internucleotide linkages. Theantisense oligonucleotide in some aspects is single-stranded and inother aspects is double-stranded. In exemplary aspects, the antisenseoligonucleotide is synthesized and in other aspects is obtained (e.g.,isolated and/or purified) from natural sources. In exemplary aspects,the antisense molecule is a phosphodiester oligonucleotide.

In alternative aspects, the antisense molecule is an antisense nucleicacid analog, e.g., comprising non-naturally-occurring nucleotides and/ornon-naturally-occurring internucleotide linkages (e.g., phosphoroamidatelinkages, phosphorothioate linkages). In exemplary aspects, theantisense nucleic acid analog comprises at least onenon-naturally-occurring nucleotide and/or non-naturally-occurringinternucleotide linkage. In exemplary aspects, the antisense nucleicacid analog comprises one or more modified nucleotides, including, butnot limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil,5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxymethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueuosine, inosine, N⁶-isopentenyladenine, 1-methylguanine, 1-methylinosine,2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine,5-methylcytosine, N-substituted adenine, 7-methylguanine,5-methylammomethyluracil, 5-methoxyaminomethyl-2-thiouracil,beta-D-mannosylqueuosine, 5′-methoxycarboxymethyluracil,5-methoxyuracil, 2-methylthio-N⁶-isopentenyladenine, uracil-5-oxyaceticacid (v), wybutoxosine, pseudouracil, queuosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine.

In exemplary aspects, the antisense nucleic acid comprisesnon-naturally-occurring nucleotides which differ from naturallyoccurring nucleotides by comprising a chemical group in place of thephosphate group. In exemplary aspects, the antisense nucleic acid analogcomprises or is a methylphosphonate oligonucleotide, which arenoncharged oligomers in which a non-bridging oxygen atom, e.g., alphaoxygen of the phosphate, is replaced by a methyl group. In exemplaryaspects, the antisense nucleic acid analog comprises or is aphosphorothioate, wherein at least one of the non-bridging oxygen atom,e.g., alpha oxygen of the phosphate, is replaced by a sulfur. Inexemplary aspects, the antisense nucleic acid analog comprises or is aboranophosphate olignucleotide, wherein at least one of the non-bridgingoxygen atom, e.g., alpha oxygen of the phosphate, is replaced by —BH₃.

In exemplary aspects, the antisense nucleic acid analog comprises atleast one non-naturally-occurring nucleotide which differs fromnaturally occurring nucleotides by comprising a ring structure otherthan ribose or 2-deoxyribose. In exemplary aspects, the antisensenucleic acid analog is an analog comprising a replacement of thehydroxyl at the 2′-position of ribose with an O-alkyl group, e.g.,—O—CH₃, —OCH₂CH₃. In exemplary aspects, the antisense nucleic acidanalog comprises a modified ribonucleotide wherein the 2′ hydroxyl ofribose is modified to methoxy (OMe) or methoxy-ethyl (MOE) group. Inexemplary aspects, the antisense nucleic acid analog comprises amodified ribonucleotide wherein the 2′ hydroxyl of ribose is replacedwith allyl, amino, azido, halo, thio, 0-allyl, O—C₁-C₁₀ alkyl, O—C₁-C₁₀substituted alkyl, O—C₁-C₁₀ alkoxy, O—C₁-C₁₀ substituted alkoxy, OCF₃,O(CH₂)₂SCH₃, O(CH₂)₂—O—N(R¹)(R²), or O(CH₂)—C(═O)—N(R¹)(R²), whereineach of R¹ and R² is independently selected from the group consisting ofH, an amino protecting group or substituted or unsubstituted C₁-C₁₀alkyl. In exemplary aspects, the antisense nucleic acid analog comprisesa modified ribonucleotide wherein the 2′ hydroxyl of ribose is replacedwith 2′F, SH, CN, OCN, CF₃, O-alkyl, S-Alkyl, N(R¹)alkyl, O-alkenyl,S-alkenyl, or N(R¹)-alkenyl, O-alkynyl, S-alkynyl, N(R¹)-alkynyl,O-alkylenyl, 0-Alkyl, alknyyl, alkaryl, aralkyl, O-alkaryl, orO-aralkyl. In exemplary aspects, the antisense nucleic acid analog is ananalog comprising a replacement of the hydrogen at the 2′-position ofribose with halo, e.g., F. In exemplary aspects, the antisense nucleicacid analog comprises a fluorine derivative nucleic acid.

In exemplary aspects, the antisense nucleic acid analog comprises asubstituted ring. In exemplary aspects, the antisense nucleic acidanalog is or comprises a hexitol nucleic acid. In exemplary aspects, theantisense nucleic acid analog is or comprises a nucleotide with abicyclic or tricyclic sugar moiety. In exemplary aspects, the bicyclicsugar moiety comprises a bridge between the 4′ and 2′ furanose ringatoms. Examplary moieties include, but are not limited to:—[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—,—C(R_(a)R_(b))—N(R)—O— or, —C(R_(a)R_(b))—O—N(R)—; 4′-CH₂-2′,4′-(CH₂)₂-2′, 4′-(CH₂)₃-2′, 4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-T;4′-(CH₂)₂—O-2′ (ENA); 4′-CH(CH₃)—O-T (cEt) and 4′-CH(CH₂OCH₃)—O-2′,4′-C(CH₃)(CH₃)—O-2′, 4′-CH₂—N(OCH₃)-2′, 4′-CH₂—O—N(CH₃)-2′4′-CH₂—O—N(R)-2′, and 4′-CH₂—N(R)—O-2′-, wherein each R is,independently, H, a protecting group, or C₁C₁₂ alkyl; 4′-CH₂—N(R)—O-2′,wherein R is H, C₁-C₁₂ alkyl, or a protecting group,4′-CH₂-C(H)(CH₃)-2′, 4′-CH₂—C(═CH₂)-2′. Such antisense nucleic acidanalogs are known in the art. See, e.g., International ApplicationPublication No. WO 2008/154401, U.S. Pat. No. 7,399,845, InternationalApplication Publication No. WO2009/006478, International ApplicationPublication No. WO2008/150729, U.S. Application Publication No.US2004/0171570, U.S. Pat. No. 7,427,672, and Chattopadhyaya, et al, J.Org. Chem.,2009, 74, 118-134). In exemplary aspects, the antisensenucleic acid analog comprises a nucleoside comprising a bicyclic sugarmoiety, or a bicyclic nucleoside (BNA). In exemplary aspects, theantisense nucleic acid analog comprises a BNA selected from the groupconsisting of: α-L-Methyleneoxy (4′-CH₂-0-2′) BNA, Aminooxy(4′-CH₂-0-N(R)-2′) BNA, β-D-Methyleneoxy (4′-CH₂-0-2′) BNA, Ethyleneoxy(4′-(CH₂)₂-0-2′) BNA, methylene-amino (4′-CH2-N(R)-2′) BNA, methylcarbocyclic (4′-CH₂—CH(CH₃)-2′) BNA, Methyl(methyleneoxy)(4′-CH(CH₃)-0-2′) BNA (also known as constrained ethyl or cEt),methylene-thio (4′-CH₂—S-2′) BNA, Oxyamino (4′-CH₂—N(R)-0-2′) BNA, andpropylene carbocyclic (4′-(CH₂)₃-2′) BNA. Such BNAs are described in theart. See, e.g., International Patent Publication No. WO 2014/071078.

In exemplary aspects, the antisense nucleic acid analog comprises amodified backbone. In exemplary aspects, the antisense nucleic acidanalog is or comprises a peptide nucleic acid (PNA) containing anuncharged flexible polyamide backbone comprising repeatingN-(2-aminoethyl)glycine units to which the nucleobases are attached viamethylene carbonyl linkers. In exemplary aspects, the antisense nucleicacid analog comprises a backbone substitution. In exemplary aspects, theantisense nucleic acid analog is or comprises an N3′→P5′phosphoramidate, which results from the replacement of the oxygen at the3′ position on ribose by an amine group. Such nucleic acid analogs arefurther described in Dias and Stein, Molec Cancer Ther 1: 347-355(2002). In exemplary aspects, the antisense nucleic acid analogcomprises a nucleotide comprising a conformational lock. In exemplaryaspects, the antisense nucleic acid analog is or comprises a lockednucleic acid.

In exemplary aspects, the antisense nucleic acid analog comprises a6-membered morpholine ring, in place of the ribose or 2-deoxyribose ringfound in RNA or DNA. In exemplary aspects, the antisense nucleic acidanalog comprises non-ionic phophorodiamidate intersubunit linkages inplace of anionic phophodiester linkages found in RNA and DNA. Inexemplary aspects, the nucleic acid analog comprises nucleobases (e.g.,adenine (A), cytosine (C), guanine (G), thymine, thymine (T), uracil(U)) found in RNA and DNA. In exemplary aspects, the IRES inhibitor is aMorpholino oligomer comprising a polymer of subunits, each subunit ofwhich comprises a 6-membered morpholine ring and a nucleobase (e.g., A,C, G, T, U), wherein the units are linked via non-ionicphophorodiamidate intersubunit linkages. For purposes herein, whenreferring to the sequence of a Morpholino oligomer, the conventionalsingle-letter nucleobase codes (e.g., A, C, G, T, U) are used to referto the nucleobase attached to the morpholine ring.

In exemplary aspects, the Morpholino oligomer binds to at least aportion of the sequence of SEQ ID NO: 5 of the α1A mRNA. In exemplaryaspects, the Morpholino oligomer binds to at least a portion of thesequence of SEQ ID NO: 6 of the α1A mRNA. In exemplary aspects, theMorpholino oligomer binds to at least a portion of the sequence of SEQID NO: 7 of the α1A mRNA. In exemplary aspects, the Morpholino oligomeris complementary to at least a portion of the sequence of SEQ ID NO: 5,SEQ ID NO: 6, or SEQ ID NO: 7. The Morpholino oligomer in some aspectsis complementary to at least 15 contiguous bases of said sequence. Inexemplary aspects, the Morpholino oligomer comprises at least 15contiguous bases of SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11. Inexemplary aspects, the Morpholino oligomer comprises at least 15contiguous bases that differ by not more than 3 bases from a portion of15 contiguous bases of SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11. Inexemplary aspects, the Morpholino oligomer comprises at least 15contiguous bases that is at least 90% identical to a portion of 15contiguous bases of SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11. Inexemplary aspects, the Morpholino oligomer comprises, consistsessentially of, or consists of SEQ ID NO: 8.

In exemplary aspects, the Morpholino oligomer comprises the sequence ofSEQ ID NO: 8. In exemplary aspects, the Morpholino oligomer comprises atleast 15 contiguous bases of a sequence that (i) differs from SEQ ID NO:8 by not more than 3 bases (e.g., not more than 4, 5, 6, 7, 8, 9, 10bases), (ii) is at least 90% (e.g., at least 93%, at least 95%, at least98%, at least 99%) identical to the sequence set forth in SEQ ID NO: 8,or (iii) is completely complementary to at least a portion of thesequence of SEQ ID NO: 6 or 7.

In exemplary aspects, the Morpholino oligomer binds to at least aportion of the sequence of SEQ ID NO: 56 of the α1A mRNA. In exemplaryaspects, the Morpholino oligomer binds to at least a portion of thesequence of SEQ ID NO: 57 of the α1A mRNA. In exemplary aspects, theMorpholino oligomer is complementary to at least a portion of thesequence of SEQ ID NO: 56 or SEQ ID NO: 57. The Morpholino oligomer insome aspects is complementary to at least 15 contiguous bases of saidsequence. In exemplary aspects, the Morpholino oligomer comprises atleast 15 contiguous bases of SEQ ID NO: 58 or SEQ ID NO: 59. Inexemplary aspects, the Morpholino oligomer comprises at least 15contiguous bases that differ by not more than 3 bases from a portion of15 contiguous bases of SEQ ID NO: 58 or SEQ ID NO: 59. In exemplaryaspects, the Morpholino oligomer comprises at least 15 contiguous basesthat is at least 90% identical to a portion of 15 contiguous bases ofSEQ ID NO: 58 or SEQ ID NO: 59. In exemplary aspects, the Morpholinooligomer comprises, consists essentially of, or consists of SEQ ID NO:55.

In exemplary aspects, the Morpholino oligomer comprises the sequence ofSEQ ID NO: 55. In exemplary aspects, the Morpholino oligomer comprisesat least 15 contiguous bases of a sequence that (i) differs from SEQ IDNO: 55 by not more than 3 bases (e.g., not more than 4, 5, 6, 7, 8, 9,10 bases), (ii) is at least 90% (e.g., at least 93%, at least 95%, atleast 98%, at least 99%) identical to the sequence set forth in SEQ IDNO: 55, or (iii) is completely complementary to at least a portion ofthe sequence of SEQ ID NO: 56 or 57.

Subjects

As used herein, the term “subject” is meant any living organism. Inexemplary aspects, the subject is a mammal. The term “mammal” as usedherein refers to any mammal, including, but not limited to, mammals ofthe order Rodentia, such as mice and hamsters, and mammals of the orderLogomorpha, such as rabbits. It is preferred that the mammals are fromthe order Carnivora, including Felines (cats) and Canines (dogs). It isfurther preferred that the mammals are from the order Artiodactyla,including Bovines (cows) and Swines (pigs) or of the orderPerssodactyla, including Equines (horses). It is further preferred thatthe mammals are of the order Primates, Ceboids, or Simoids (monkeys) orof the order Anthropoids (humans and apes). In exemplary aspect, thesubject is a human.

In exemplary embodiments, the human has, is diagnosed with, and/orsuffers from a genetic disease, including, any of those known in the artand/or described herein. In exemplary aspects, the subject has, isdiagnosed with, and/or suffers from a trinucleotide repeat disorder,such as a polyglutamine disease. In exemplary aspects, the human has, isdiagnosed with, and/or suffers from SCA6.

Vectors

In exemplary aspects, the IRES inhibitor is a recombinant expressionvector encoding an antisense molecule described herein. Accordingly, theinvention provides a recombinant expression vector comprising anucleotide sequence encoding an antisense molecule described herein. Forpurposes herein, the term “recombinant expression vector” means agenetically-modified oligonucleotide or polynucleotide construct thatpermits the expression of a nucleic acid by a host cell, when theconstruct comprises a nucleotide sequence encoding the nucleic acid, andthe vector is contacted with the cell under conditions sufficient tohave the nucleic acid expressed within the cell. The recombinantexpression vectors of the invention are not naturally-occurring as awhole. However, parts of the vectors may be naturally-occurring. Theinventive recombinant expression vectors may comprise any type ofnucleotides, including, but not limited to DNA and RNA, which may besingle-stranded or double-stranded, synthesized or obtained in part fromnatural sources, and which may contain natural, non-natural or alterednucleotides. The recombinant expression vectors may comprisenaturally-occurring or non-naturally-occurring internucleotide linkages,or both types of linkages. In exemplary aspects, the altered nucleotidesor non-naturally occurring internucleotide linkages do not hinder thetranscription or replication of the vector.

In exemplary aspects, the recombinant expression vector encodes anantisense molecule which binds to SEQ ID NO: 180. In exemplary aspects,the recombinant expression vector encodes an antisense molecule whichbinds to at least a portion of the sequence of SEQ ID NO: 180 of the α1AmRNA, wherein the portion is at least 5 contiguous nucleotides (or atleast 2 or at least 3 contiguous nucleotides) of SEQ ID NO: 180. Inexemplary aspects, the recombinant expression vector encodes anantisense molecule which binds to at least 15 contiguous nucleotides ofthe sequence of SEQ ID NO: 180. In exemplary aspects, the recombinantexpression vector encodes an antisense molecule which binds to at least20 contiguous nucleotides of the sequence of SEQ ID NO: 180. Inexemplary aspects, the recombinant expression vector encodes anantisense molecule binds to at least 25 contiguous nucleotides of thesequence of SEQ ID NO: 180.

In exemplary aspects, the recombinant expression vector encodes anantisense molecule which is complementary to at least a portion of thesequence of SEQ ID NO: 180. In exemplary aspects, the recombinantexpression vector encodes an antisense molecule which is complementaryto at least 15 contiguous bases of said sequence of SEQ ID NO: 180. Inexemplary aspects, the recombinant expression vector encodes anantisense molecule which is complementary to at least 20 contiguousbases of the sequence of SEQ ID NO: 180. In exemplary aspects, therecombinant expression vector encodes an antisense molecule which iscomplementary to at least 25 contiguous bases of the sequence of SEQ IDNO: 180. In exemplary aspects, the recombinant expression vector encodesan antisense oligonucleotide comprising at least 15 contiguous bases ofSEQ ID NO: 179. In exemplary aspects, the recombinant expression vectorencodes an antisense oligonucleotide comprising at least 15 contiguousbases that differs by not more than 3 bases from a portion of 15contiguous bases of SEQ ID NO: 179. In exemplary aspects, therecombinant expression vector encodes an antisense oligonucleotidecomprising at least 15 contiguous bases that is at least 90% identicalto a portion of 15 contiguous bases of SEQ ID NO: 179. In exemplaryaspects, the recombinant expression vector encodes an antisenseoligonucleotide comprising, consisting essentially of, or consisting ofSEQ ID NO: 179.

In exemplary aspects, the recombinant expression vector encodes an miRNAwhich targets a portion of the IRES of the CACNA1A gene wherein theportion comprises sequence of SEQ ID NO: 180. As understood by theordinarily skilled artisan, miRNA does not require perfect pairing toits target. In exemplary aspects, the recombinant expression vectorencodes an miRNA which binds to at least a portion of the sequence ofSEQ ID NO: 180. In exemplary aspects, the recombinant expression vectorencodes an miRNA which is complementary to at least a portion of thesequence of SEQ ID NO: 180. The recombinant expression vector encodes anmiRNA which in some aspects is complementary to at least 15 contiguousbases of said sequence. In exemplary aspects, the recombinant expressionvector encodes an miRNA which comprises at least 15 contiguous bases ofSEQ ID NO: 179. In exemplary aspects, the recombinant expression vectorencodes an miRNA which comprises at least 15 contiguous bases thatdiffer by not more than 3 bases from a portion of 15 contiguous bases ofSEQ ID NO: 179. In exemplary aspects, the recombinant expression vectorencodes an miRNA which comprises at least 15 contiguous bases that is atleast 90% identical to a portion of 15 contiguous bases of SEQ ID NO:179. In exemplary aspects, the recombinant expression vector encodes anmiRNA which comprises, consists essentially of, or consists of SEQ IDNO: 179. In exemplary aspects, the recombinant expression vector encodesan miRNA is about 15, about 16, about 17, about 18, about 19, about 20,about 21, about 22, about 23, about 24 nt in length.

In exemplary aspects, the recombinant expression vector encodes an miRNAcomprising the sequence of SEQ ID NO: 179. In exemplary aspects, therecombinant expression vector encodes an miRNA comprising at least 15contiguous bases of a sequence that (i) differs from SEQ ID NO: 179 bynot more than 3 bases (e.g., not more than 4, 5, 6, 7, 8, 9, 10 bases),(ii) is at least 90% (e.g., at least 93%, at least 95%, at least 98%, atleast 99%) identical to the sequence set forth in SEQ ID NO: 179, or(iii) is completely complementary to at least a portion of the sequenceof SEQ ID NO: 180.

In exemplary aspects, the recombinant expression vector encodes anantisense molecule which is a precursor of miRNA comprising SEQ ID NO:179. In exemplary aspects, the recombinant expression vector encodes anantisense molecule which is a primary miRNA precursor (pri-miRNA)comprising SEQ ID NO: 179 which is optionally, capped, polyadenylated,and/or comprises double-stranded stem-loop structures. In exemplaryaspects, the recombinant expression vector encodes an antisense moleculewhich is a pre-miRNA precursor comprising SEQ ID NO: 179, which isoptionally about 70 to about 100 nt long and/or comprises a hairpinstructure.

In exemplary aspects, the recombinant expression vector comprises anucleotide sequence of SEQ ID NO: 181. In exemplary aspects, therecombinant expression vector comprises a nucleotide sequence whichcomprises at least 15 contiguous bases of SEQ ID NO: 181. For example,the recombinant expression vector comprises a nucleotide sequence whichcomprises at least or about 16 contiguous bases of SEQ ID NO: 181, atleast or about 17 contiguous bases of SEQ ID NO: 181, at least or about18 contiguous bases of SEQ ID NO: 181, at least or about 19 contiguousbases of SEQ ID NO: 181, at least or about 20 contiguous bases of SEQ IDNO: 181, at least or about 21 contiguous bases of SEQ ID NO: 181, atleast or about 22 contiguous bases of SEQ ID NO: 181, at least or about23 contiguous bases of SEQ ID NO: 181, at least or about 24 contiguousbases of SEQ ID NO: 181, at least or about 25 contiguous bases of SEQ IDNO: 181, at least or about 26 contiguous bases of SEQ ID NO: 181, atleast or about 27 contiguous bases of SEQ ID NO: 181, or at least orabout 28 contiguous bases of SEQ ID NO: 181. In exemplary aspects, therecombinant expression vector comprises a nucleotide sequence thatdiffers from SEQ ID NO: 181 by not more than 3 bases (e.g., not morethan 4, 5, 6, 7, 8, 9, 10 bases). In exemplary aspects, the recombinantexpression vector comprises a nucleotide sequence which is at least 90%(e.g., at least 93%, at least 95%, at least 98%, at least 99%) identicalto the sequence of SEQ ID NO: 181.

The recombinant expression vector of the invention may be any suitablerecombinant expression vector, and may be used to transform or transfectany suitable host or host cell. Suitable vectors include those designedfor propagation and expansion or for expression or both, such asplasmids and viruses. The vector may be selected from the groupconsisting of the pUC series (Fermentas Life Sciences), the pBluescriptseries (Stratagene, LaJolla, Calif.), the pET series (Novagen, Madison,Wis.), the pGEX series (Pharmacia Biotech, Uppsala, Sweden), and the pEXseries (Clontech, Palo Alto, Calif.). Bacteriophage vectors, such asλGTIO, λGT11, λZapII (Stratagene), λEMBL4, and, λNMI 149, also may beused. Examples of plant expression vectors include pBIO1, pBI101.2,pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animal expressionvectors include pEUK-Cl, pMAM and pMAMneo (Clontech).

In exemplary aspects, the recombinant expression vector is a viralvector, e.g., a retroviral vector. In exemplary aspects, the recombinantexpression vector is a cytoplasmic RNA viral vector. In exemplaryaspects, the recombinant expression vector is k-borne encephalitis viralvector or a Sindbis viral vector. In exemplary aspects, the recombinantexpression vector is an influenza A viral vector, an adenoviral vector,an adeno-associated vector (AAV), a lentiviral vector, an arboviralvector, an Epstein Barr viral vector, a flavivirus tick-borneencephalitis viral vector, a bovine leukemia viral vector, an humanimmunodeficiency viral vector, a simian immunodeficiency viral vector, avesicular stomatitis viral vector, herpes simplex viral vector, poxviral vector, parvoviral vector, vaccinia viral vector, agammaretroviral vector, or an alphaviral vector. In exemplary aspects,the AAV is a ssAAV8, scAAV8, ssAAV9, scAAV7, scAAV9, ssAAV2, ssAAV6,ssAAV1, ssAAV1/2, ssAAV5. In exemplary aspects, the arboviral vector isa flaviviral vector, togaviral vector, bunyaviral vector, rhabdoviralvector, or a reoviral vector. Such vectors are known in the art. See,e.g., Peng et al., Adv Drug Deliv Rev 88: 108-122 (2015); Borel et al.,Mol Ther 22(4): 692-701 (2014); Usme-Ciro et al., Virol J 10: 185(2013); Khatri et al., Crit Rev Ther Drug Carrier Syst 29(6): 487-527(2012); Liu et al., Biochim Biophs Acta 1809(11-12): 732-745 (2011); Kayet al., Nature 7(1): 33-40 (2001); and Asgari, Viruses 6(9): 3514-3514(2014).

In exemplary aspects, the recombinant expression vector comprises atleast one element from a viral vector. In exemplary aspects, therecombinant expression vector comprises a combination of elements fromdifferent viral vectors.

In exemplary aspects, the recombinant expression vector is a non-viralsynthetic vector, including any of those described in Wang et al., AdvDrug Deliv Rev 81: 142-160 (2015).

The recombinant expression vectors of the invention may be preparedusing standard recombinant DNA techniques described in, for example,Sambrook et al., supra, and Ausubel et al., supra. Constructs ofexpression vectors, which are circular or linear, may be prepared tocontain a replication system functional in a prokaryotic or eukaryotichost cell. Replication systems may be derived, e.g., from CoIEl, 2μplasmid, k, SV40, bovine papilloma virus, and the like.

In exemplary aspects, the recombinant expression vector comprisesregulatory sequences, such as transcription and translation initiationand termination codons, which are specific to the type of host (e.g.,bacterium, fungus, plant, or animal) into which the vector is to beintroduced, as appropriate and taking into consideration whether thevector is DNA- or RNA-based.

The recombinant expression vector may include one or more markers ormarker genes, which allow for selection of transformed or transfectedhosts. Marker genes include biocide resistance, e.g., resistance toantibiotics, heavy metals, etc., complementation in an auxotrophic hostto provide prototrophy, and the like. Suitable markers and marker genesfor the inventive expression vectors include, for instance, His-tags,FLAG tags, green fluorescence protein genes, red fluorescence proteingenes, neomycin/G418 resistance genes, hygromycin resistance genes,histidinol resistance genes, tetracycline resistance genes, andampicillin resistance genes. In exemplary aspects, the recombinantexpression vector comprises a nucleotide sequence encoding greenfluorescent protein (GFP) or another fluorescent protein (e.g., redfluorescent protein (RFP), cyan fluorescent protein (CFP), yellowfluorescent protein, orange fluorescent protein, far red fluorescentprotein, and the like).

The recombinant expression vector may comprise a native or non-nativepromoter operably linked to the nucleotide sequence encoding theantisense molecule. The selection of promoters, e.g., strong, weak,inducible, tissue-specific and developmental-specific, is within theordinary skill of the artisan. In exemplary aspects, the recombinantexpression vector comprises a non-native promoter. In exemplary aspects,the recombinant expression vector comprises a promoter which is notfound upstream or near the antisense molecule in its naturalenvironment. In exemplary aspects, the recombinant expression vectorcomprises a non-human promoter. The promoter in exemplary aspects is anon-viral promoter or a viral promoter, e.g., a cytomegalovirus (CMV)promoter, an SV40 promoter, an RSV promoter, and a promoter found in thelong-terminal repeat of the murine stem cell virus.

In exemplary aspects, the vector is a recombinant adeno-associated viral(AAV) vector encoding an antisense molecule comprising an nucleotidesequence (e.g., an antisense molecule) which binds to a portion of anIRES of a CACNA1A gene comprising the sequence of SEQ ID NO: 180 thesequence of SEQ ID NO: 179. In exemplary aspects, the AAV vector is anAAV serotype 9 (AAV9) vector. In exemplary aspects, the recombinant AAVvector comprises one or more of a promoter, a pair of inverted terminalrepeats (ITRs), and a polyadenylation signal sequence. In exemplaryaspects, the promoter is a human cytomegalovirus (CMV) immediate earlypromoter. In exemplary aspects, the ITRs are AAV ITRs. In exemplaryaspects, the polyadenylation signal sequence is an simian virus 40(SV40) polyadenylation signal sequence. In exemplary aspects, therecombinant expression vector comprises a woodchuck hepatitis virusposttranscriptional regulatory element (WPRE). In exemplary aspects, therecombinant expression vector comprises an ITR upstream of the human CMVimmediate early promoter, which is located upstream of the nucleotidesequence encoding the antisense molecule, which is located upstream ofthe WPRE which is located upstream of the SV40 polyadenylation signalsequence which is located upstream of the the other ITR.

The inventive recombinant expression vectors may be designed for eithertransient expression, for stable expression, or for both. Also, therecombinant expression vectors may be made for constitutive expressionor for inducible expression. Further, the recombinant expression vectorsmay be made to include a suicide gene. In exemplary aspects, theinventive recombinant expression vectors are designed for targetedexpression. In exemplary aspects, the recombinant expression vectorcomprises one or more elements which cause the antisense molecule to beexpressed by a specific cell population.

As used herein, the term “suicide gene” refers to a gene that causes thecell expressing the suicide gene to die. The suicide gene may be a genethat confers sensitivity to an agent, e.g., a drug, upon the cell inwhich the gene is expressed, and causes the cell to die when the cell iscontacted with or exposed to the agent. Suicide genes are known in theart (see, for example, Suicide Gene Therapy: Methods and Reviews.Springer, Caroline J. (Maycer Research UK Centre for Maycer Therapeuticsat the Institute of Maycer Research, Sutton, Surrey, UK), Humana Press,2004) and include, for example, the Herpes Simplex Virus (HSV) thymidinekinase (TK) gene, cytosine daminase, purine nucleoside phosphorylase,and nitroreductase.

Host Cells

In exemplary aspects, the IRES inhibitor is a cell (e.g. a host cell),or a population of cells, expressing an antisense molecule describedherein. In exemplary aspects, the IRES inhibitor is a host cell, or apopulation of host cells, comprising a recombinant expression vectorencoding any of the antisense molecules described herein. Providedherein is a host cell, or a population of cells, comprising an antisensemolecule described herein or an extracellular vesicle described herein.Provided herein is a cell, or a population of cells, expressing anantisense molecule described herein. Provided herein is a cell, or apopulation of cells, comprising a recombinant expression vectordescribed herein. In exemplary aspects, the cell or population of cellsis genetically engineered to comprise and express any one of theantisense molecules or recombinant expression vectors described herein.As used herein, the term “host cell” refers to any type of cell that maycontain the antisense molecule or vector described herein. In exemplaryaspects, the host cell is a eukaryotic cell, e.g., plant, animal, fungi,or algae, or may be a prokaryotic cell, e.g., bacteria or protozoa. Inexemplary aspects, the host cells is a cell originating or obtained froma subject, as described herein. In exemplary aspects, the host celloriginates from or is obtained from a mammal. As used herein, the term“mammal” refers to any mammal, including, but not limited to, mammals ofthe order Rodentia, such as mice and hamsters, and mammals of the orderLogomorpha, such as rabbits. It is preferred that the mammals are fromthe order Carnivora, including Felines (cats) and Canines (dogs). It ismore preferred that the mammals are from the order Artiodactyla,including Bo vines (cows) and S wines (pigs) or of the orderPerssodactyla, including Equines (horses). It is most preferred that themammals are of the order Primates, Ceboids, or Simoids (monkeys) or ofthe order Anthropoids (humans and apes). An especially preferred mammalis the human.

In exemplary aspects, the host cell is a cultured cell or a primarycell, i.e., isolated directly from an organism, e.g., a human. The hostcell in exemplary aspects is an adherent cell or a suspended cell, i.e.,a cell that grows in suspension. Suitable host cells are known in theart and include, for instance, DH5α E. coli cells, Chinese hamsterovarian (CHO) cells, monkey VERO cells, T293 cells, COS cells, HEK293cells, and the like. For purposes of amplifying or replicating therecombinant expression vector, the host cell is preferably a prokaryoticcell, e.g., a DH5a cell. In exemplary aspects, the host cell is a humancell. The host cell may be of any cell type, may originate from any typeof tissue, and may be of any developmental stage.

In exemplary aspects, the host cell is a mammalian, e.g., human, stemcell. In exemplary aspects, the host cell is a mammalian, e.g., human,cell of the nervous system.

Also provided by the invention is a population of cells comprising atleast one host cell described herein. The population of cells may be aheterogeneous population comprising the host cell comprising any of theexpression vectors described, in addition to at least one other cell,e.g., a host cell, which does not comprise any of the recombinantexpression vectors. Alternatively, the population of cells may be asubstantially homogeneous population, in which the population comprisesmainly of host cells (e.g., consisting essentially of) comprising theexpression vector. The population also may be a clonal population ofcells, in which all cells of the population are clones of a single hostcell comprising a recombinant expression vector, such that all cells ofthe population comprise the recombinant expression vector. In exemplaryembodiments of the invention, the population of cells is a clonalpopulation comprising host cells expressing a nucleic acid or a vectordescribed herein.

Delivery Systems

In exemplary aspects, the antisense molecule is part of a deliverysystem, e.g., a non-viral delivery system. Delivery systems suitable foruse in the methods of the present inventions and provided herein includeany of those known in the art. See, e.g., Zhang et al., 2013, supra. Inexemplary aspects, the delivery system is a liposome, an aptamercomplex, a nanoparticle, or a dendrimer. Accordingly, the inventionprovides such delivery systems. The invention provides a liposome, anaptamer complex, a nanoparticle, a dendrimer, or an extracellularvesicle comprising an antisense molecule or a recombinant expressionvector described herein.

In exemplary aspects, the antisense molecule is part of anaptamer-oligonucleotide conjugate. Such conjugates are described in theart. See, e.g., Liu et al., Cancer investigation 30:577-582 (2012). Inexemplary aspects, the antisense molecule is part of a lipid-baseddelivery system, such as siPORT, MaxSuppressor, LipoTrust, each of whichare commercially-available and described in the art. See, e.g., Wu etal., Molecular Pharmaceutics 8:1381-1389 (2011); Craig et al., Leukemia26:2421-2424 (2012); Trang et al., Mol Ther 19:1116-1122 (2011); Akao etal., Cancer Gene Ther 17:398-408 (2010). In exemplary aspects, theantisense molecule is part of a lipid-based delivery system that is notcommercially available, e.g., a 98N12-5 delivery system (Akinc et al.,Nature Biotechnology 26:561-569 (2008); a1,2-Di-O-octadecenyl-3-trimethylammonium propane(DOTMA):cholesterol:D-alpha-tocopheryl polyethylene glycol 1000succinate (TPGS) lipoplex (Wu et al., Molecular Pharmaceutics8:1381-1389 (2011); a dimethyldioctadecylammonium bromide(DDAB):cholesterol:TPGS lipoplex (Piao et al., Molec Therapy20:1261-1269 (2012); a targeted liposome-hyaluronic acid (LPH)nanoparticle (Chen et al., Mole Ther 18:1650-1656 (2010); and Liu etal., Molec Pharmaceutics 8:250-259 (2011)); an nanotransporterinterfering nanoparticle-7 (iNOP-7) (Su et al., Nucleic Acids Res 39:e38(2011)); a DC-6-140-DOPE-cholesterol liposome (Rai et al., Molec CancerTher 10:1720-1727 (2011)); or a solid lipid nanoparticle (Shi et al.,Systemic Delivery of microRNA-34A for Cancer Stem Cell Therapy,Angewandte Chemie (2013)).

In exemplary aspects, the antisense molecule is part of apolyethyleneimine (PEI) conjugate (Ibrahim et al., Cancer Research71:5214-5224 (2011)); a polyurethane-short brance PEI conjugate (Chiouet al., J Control Release 159:240-250 (2012); a dendrimer (e.g., apoly(amidoamine) dendrimer (Ren et al., BMC Cancer 10:27 (2010); Ren etal., J Biomater Sci Polym Ed 21:303-314 (2010)); apoly(lactide-co-glycolide) (PLGA) nanoparticle (Cheng et al., MolecPharm 9:1481-1488 (2012); Babar et al., PNAS e1695-e1704 (2012)), adisialoganglioside-targeting silica nanoparticle or a modifiedultrasmall magnetic nanoparticle (Tivnan et al., PLoS One 7:e38129(2012); Yigit et al., Oncogene 32, 1530-1538 (2013)).

In exemplary aspects, the antisense molecule is part of an extracellularvesicle, such as e.g., an exosome. Such vesicles for delivery of smallRNA molecules are described in Hagiwara et al., Drug Deliv and TranslRes 4: 31-37 (2014); Momen-Heravi et al., Nature Scientific Reports 5:09991 (2015); Wang et al., Asian PAc J Cancer Prev 16(10): 4203-4209(2015); and International Application Publication No. WO/2014/028763;and include any vesicle in the extracellular space. In exemplaryaspects, the extracellular vesicle is an exosome. In exemplary aspects,the exosome is 40-100 nm in diameter and are derived frommultivescicular endosomes. In exemplary aspects, the extracellularvesicle is a microvesicle. In exemplary aspects, the microvesicle is50-1000 nm in diameter and is generated by budding at the plasmamembrane.

The present invention provides a liposome, aptamer complex,nanoparticle, dendrimer, or extracellular vesicle comprising anantisense molecule, e.g., an miRNA, which targets a portion of the IRESof the CACNA1A gene wherein the portion comprises sequence of SEQ ID NO:180. In exemplary aspects, the liposome, aptamer complex, nanoparticle,dendrimer, or extracellular vesicle comprises an antisense molecule,e.g., miRNA, which binds to at least a portion of the sequence of SEQ IDNO: 180. In exemplary aspects, the liposome, aptamer complex,nanoparticle, dendrimer, or extracellular vesicle comprises an antisensemolecule, e.g., an miRNA, which is complementary to at least a portionof the sequence of SEQ ID NO: 180. The antisense molecule, e.g., miRNA,in some aspects is complementary to at least 15 contiguous bases of saidsequence. In exemplary aspects, the liposome, aptamer complex,nanoparticle, dendrimer, or extracellular vesicle comprises an antisensemolecule, e.g., miRNA, which comprises at least 15 contiguous bases ofSEQ ID NO: 179. In exemplary aspects, the liposome, aptamer complex,nanoparticle, dendrimer, or extracellular vesicle comprises an antisensemolecule, e.g., miRNA, which comprises at least 15 contiguous bases thatdiffer by not more than 3 bases from a portion of 15 contiguous bases ofSEQ ID NO: 179. In exemplary aspects, the liposome, aptamer complex,nanoparticle, dendrimer, or extracellular vesicle comprises an antisensemolecule, e.g., miRNA, which comprises at least 15 contiguous bases thatis at least 90% identical to a portion of 15 contiguous bases of SEQ IDNO: 179. In exemplary aspects, the liposome, aptamer complex,nanoparticle, dendrimer, or extracellular vesicle comprises an antisensemolecule, e.g., miRNA, which comprises, consists essentially of, orconsists of SEQ ID NO: 179. In exemplary aspects, the miRNA is about 15,about 16, about 17, about 18, about 19, about 20, about 21, about 22,about 23, about 24 nt in length.

In exemplary aspects, the liposome, aptamer complex, nanoparticle,dendrimer, or extracellular vesicle comprises an antisense molecule,which is a precursor of miRNA comprising SEQ ID NO: 179. In exemplaryaspects, the antisense molecule is a primary miRNA precursor (pri-miRNA)comprising SEQ ID NO: 179 which is optionally, capped, polyadenylated,and/or comprises double-stranded stem-loop structures. In exemplaryaspects, the liposome, aptamer complex, nanoparticle, dendrimer, orextracellular vesicle comprises an antisense molecule, which is apre-miRNA precursor comprising SEQ ID NO: 179, which is optionally about70 to about 100 nt long and/or comprises a hairpin structure.

In exemplary aspects, the liposome, aptamer complex, nanoparticle,dendrimer, or extracellular vesicle comprises an antisense molecule,e.g., miRNA, comprising the sequence of SEQ ID NO: 179. In exemplaryaspects, the the liposome, aptamer complex, nanoparticle, dendrimer, orextracellular vesicle comprises an antisense molecule, e.g., miRNA,comprising at least 15 contiguous bases of a sequence that (i) differsfrom SEQ ID NO: 179 by not more than 3 bases (e.g., not more than 4, 5,6, 7, 8, 9, 10 bases), (ii) is at least 90% (e.g., at least 93%, atleast 95%, at least 98%, at least 99%) identical to the sequence setforth in SEQ ID NO: 179, or (iii) is completely complementary to atleast a portion of the sequence of SEQ ID NO: 180.

In exemplary aspects, the liposome, aptamer complex, nanoparticle,dendrimer, or extracellular vesicle comprises a recombinant expressionvector comprising a nucleotide sequence of SEQ ID NO: 181. In exemplaryaspects, the liposome, aptamer complex, nanoparticle, dendrimer, orextracellular vesicle comprises a recombinant expression vectorcomprising a nucleotide sequence which comprises at least 15 contiguousbases of SEQ ID NO: 181. For example, the recombinant expression vectorcomprises a nucleotide sequence which comprises at least or about 16contiguous bases of SEQ ID NO: 181, at least or about 17 contiguousbases of SEQ ID NO: 181, at least or about 18 contiguous bases of SEQ IDNO: 181, at least or about 19 contiguous bases of SEQ ID NO: 181, atleast or about 20 contiguous bases of SEQ ID NO: 181, at least or about21 contiguous bases of SEQ ID NO: 181, at least or about 22 contiguousbases of SEQ ID NO: 181, at least or about 23 contiguous bases of SEQ IDNO: 181, at least or about 24 contiguous bases of SEQ ID NO: 181, atleast or about 25 contiguous bases of SEQ ID NO: 181, at least or about26 contiguous bases of SEQ ID NO: 181, at least or about 27 contiguousbases of SEQ ID NO: 181, or at least or about 28 contiguous bases of SEQID NO: 181. In exemplary aspects, the liposome, aptamer complex,nanoparticle, dendrimer, or extracellular vesicle comprises arecombinant expression vector comprising a nucleotide sequence thatdiffers from SEQ ID NO: 181 by not more than 3 bases (e.g., not morethan 4, 5, 6, 7, 8, 9, 10 bases). In exemplary aspects, the liposome,aptamer complex, nanoparticle, dendrimer, or extracellular vesiclecomprises a recombinant expression vector comprising a nucleotidesequence which is at least 90% (e.g., at least 93%, at least 95%, atleast 98%, at least 99%) identical to the sequence of SEQ ID NO: 181.

Antisense Molecules and Pharmaceutical Compositions Comprising the Same

The invention also provides any of the aforementioned IRES inhibitors.In this regard, the invention provides any of the aforementionedantisense molecules, e.g., antisense oligonucleotides, antisense nucleicacid analogs, suitable for use in the inventive methods. For purposesherein, in some aspects, the IRES inhibitor, e.g., antisense molecule,is isolated, purified, or not naturally-occurring or synthetic. The term“isolated” as used herein means having been removed from its naturalenvironment. The term “purified” as used herein means having beenincreased in purity, wherein “purity” is a relative term, and not to benecessarily construed as absolute purity. As used herein, the term “notnaturally-occurring” refers to a molecule or compound which is not foundin nature or is non-natural. An IRES inhibitor which is notnaturally-occurring is “non-naturally occurring.” In exemplary aspects,an IRES inhibitor which is not naturally-occurring comprises at leastone component which is not found in nature. The non-natural IRESinhibitor may comprise one or more naturally-occurring components butcomprises at least one component which is not found in nature. Thenon-naturally occurring IRES inhibitor in some aspects comprises onlynaturally occurring components, but the overall structure or arrangementof the components is not found in nature. It is preferred that noinsertions, deletions, inversions, and/or substitutions are present inthe antisense molecules of the invention. However, it may be suitable tocomprise one or more insertions, deletions, inversions, and/orsubstitutions. In exemplary aspects, the antisense molecule comprises adetectable label, such as, for instance, a radioisotope, a fluorophore,or an element particle.

The antisense molecules of the invention can be constructed based onchemical synthesis and/or enzymatic ligation reactions using proceduresknown in the art. See, for example, Sambrook et al., Molecular Cloning:A Laboratory Manual, 3^(rd) Ed., Cold Spring Harbor Press, Cold SpringHarbor, N. Y. (2001) and Ausubel et al., Current Protocols in MolecularBiology, Greene Publishing Associates and John Wiley & Sons, New York,N. Y. (1994). For example, an antisense molecule can be chemicallysynthesized using naturally occurring nucleotides or variously modifiednucleotides designed to increase the biological stability of themolecules or to increase the physical stability of the duplex formedupon hybridization (e.g., phosphorothioate derivatives and acridinesubstituted nucleotides).

In exemplary aspects, the IRES inhibitor, e.g., antisense molecule, isformulated with one or more pharmaceutically acceptable carriers,diluents, and/or excipients and is provided as part of a pharmaceuticalcomposition. In this regard, the invention further provides apharmaceutical composition comprising any of the IRES inhibitors, e.g.,antisense molecules, described herein. The pharmaceutical compositioncomprises one or more pharmaceutically acceptable carriers, diluents,and/or excipients, and is preferably sterile.

Depending on the route of administration, the particular IRES inhibitorintended for use, as well as other factors, the pharmaceuticalcomposition may comprise additional pharmaceutically acceptableingredients, including, for example, acidifying agents, additives,adsorbents, aerosol propellants, air displacement agents, alkalizingagents, anticaking agents, anticoagulants, antimicrobial preservatives,antioxidants, antiseptics, bases, binders, buffering agents, chelatingagents, coating agents, coloring agents, desiccants, detergents,diluents, disinfectants, disintegrants, dispersing agents, dissolutionenhancing agents, dyes, emollients, emulsifying agents, emulsionstabilizers, fillers, film forming agents, flavor enhancers, flavoringagents, flow enhancers, gelling agents, granulating agents, humectants,lubricants, mucoadhesives, ointment bases, ointments, oleaginousvehicles, organic bases, pastille bases, pigments, plasticizers,polishing agents, preservatives, sequestering agents, skin penetrants,solubilizing agents, solvents, stabilizing agents, suppository bases,surface IRES inhibitors, surfactants, suspending agents, sweeteningagents, therapeutic agents, thickening agents, tonicity agents, toxicityagents, viscosity-increasing agents, water-absorbing agents,water-miscible cosolvents, water softeners, or wetting agents.

Accordingly, in some embodiments, the pharmaceutical compositioncomprises any one or a combination of the following components: acacia,acesulfame potassium, acetyltributyl citrate, acetyltriethyl citrate,agar, albumin, alcohol, dehydrated alcohol, denatured alcohol, dilutealcohol, aleuritic acid, alginic acid, aliphatic polyesters, alumina,aluminum hydroxide, aluminum stearate, amylopectin, a-amylose, ascorbicacid, ascorbyl palmitate, aspartame, bacteriostatic water for injection,bentonite, bentonite magma, benzalkonium chloride, benzethoniumchloride, benzoic acid, benzyl alcohol, benzyl benzoate, bronopol,butylated hydroxyanisole, butylated hydroxytoluene, butylparaben,butylparaben sodium, calcium alginate, calcium ascorbate, calciumcarbonate, calcium cyclamate, dibasic anhydrous calcium phosphate,dibasic dehydrate calcium phosphate, tribasic calcium phosphate, calciumpropionate, calcium silicate, calcium sorbate, calcium stearate, calciumsulfate, calcium sulfate hemihydrate, canola oil, carbomer, carbondioxide, carboxymethyl cellulose calcium, carboxymethyl cellulosesodium, β-carotene, carrageenan, castor oil, hydrogenated castor oil,cationic emulsifying wax, cellulose acetate, cellulose acetatephthalate, ethyl cellulose, microcrystalline cellulose, powderedcellulose, silicified microcrystalline cellulose, sodium carboxymethylcellulose, cetostearyl alcohol, cetrimide, cetyl alcohol, chlorhexidine,chlorobutanol, chlorocresol, cholesterol, chlorhexidine acetate,chlorhexidine gluconate, chlorhexidine hydrochloride,chlorodifluoroethane (HCFC), chlorodifluoromethane, chlorofluorocarbons(CFC)chlorophenoxyethanol, chloroxylenol, corn syrup solids, anhydrouscitric acid, citric acid monohydrate, cocoa butter, coloring agents,corn oil, cottonseed oil, cresol, m-cresol, o-cresol, p-cresol,croscarmellose sodium, crospovidone, cyclamic acid, cyclodextrins,dextrates, dextrin, dextrose, dextrose anhydrous, diazolidinyl urea,dibutyl phthalate, dibutyl sebacate, diethanolamine, diethyl phthalate,difluoroethane (HFC), dimethyl-β-cyclodextrin, cyclodextrin-typecompounds such as Captisol®, dimethyl ether, dimethyl phthalate,dipotassium edentate, disodium edentate, disodium hydrogen phosphate,docusate calcium, docusate potassium, docusate sodium, dodecyl gallate,dodecyltrimethylammonium bromide, edentate calcium disodium, edtic acid,eglumine, ethyl alcohol, ethylcellulose, ethyl gallate, ethyl laurate,ethyl maltol, ethyl oleate, ethylparaben, ethylparaben potassium,ethylparaben sodium, ethyl vanillin, fructose, fructose liquid, fructosemilled, fructose pyrogen-free, powdered fructose, fumaric acid, gelatin,glucose, liquid glucose, glyceride mixtures of saturated vegetable fattyacids, glycerin, glyceryl behenate, glyceryl monooleate, glycerylmonostearate, self-emulsifying glyceryl monostearate, glycerylpalmitostearate, glycine, glycols, glycofurol, guar gum,heptafluoropropane (HFC), hexadecyltrimethylammonium bromide, highfructose syrup, human serum albumin, hydrocarbons (HC), dilutehydrochloric acid, hydrogenated vegetable oil, type II, hydroxyethylcellulose, 2-hydroxyethyl-β-cyclodextrin, hydroxypropyl cellulose,low-substituted hydroxypropyl cellulose, 2-hydroxypropyl-β-cyclodextrin,hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate,imidurea, indigo carmine, ion exchangers, iron oxides, isopropylalcohol, isopropyl myristate, isopropyl palmitate, isotonic saline,kaolin, lactic acid, lactitol, lactose, lanolin, lanolin alcohols,anhydrous lanolin, lecithin, magnesium aluminum silicate, magnesiumcarbonate, normal magnesium carbonate, magnesium carbonate anhydrous,magnesium carbonate hydroxide, magnesium hydroxide, magnesium laurylsulfate, magnesium oxide, magnesium silicate, magnesium stearate,magnesium trisilicate, magnesium trisilicate anhydrous, malic acid,malt, maltitol, maltitol solution, maltodextrin, maltol, maltose,mannitol, medium chain triglycerides, meglumine, menthol,methylcellulose, methyl methacrylate, methyl oleate, methylparaben,methylparaben potassium, methylparaben sodium, microcrystallinecellulose and carboxymethylcellulose sodium, mineral oil, light mineraloil, mineral oil and lanolin alcohols, oil, olive oil, monoethanolamine,montmorillonite, octyl gallate, oleic acid, palmitic acid, paraffin,peanut oil, petrolatum, petrolatum and lanolin alcohols, pharmaceuticalglaze, phenol, liquified phenol, phenoxyethanol, phenoxypropanol,phenylethyl alcohol, phenylmercuric acetate, phenylmercuric borate,phenylmercuric nitrate, polacrilin, polacrilin potassium, poloxamer,polydextrose, polyethylene glycol, polyethylene oxide, polyacrylates,polyethylene-polyoxypropylene-block polymers, polymethacrylates,polyoxyethylene alkyl ethers, polyoxyethylene castor oil derivatives,polyoxyethylene sorbitol fatty acid esters, polyoxyethylene stearates,polyvinyl alcohol, polyvinyl pyrrolidone, potassium alginate, potassiumbenzoate, potassium bicarbonate, potassium bisulfite, potassiumchloride, postassium citrate, potassium citrate anhydrous, potassiumhydrogen phosphate, potassium metabisulfite, monobasic potassiumphosphate, potassium propionate, potassium sorbate, povidone, propanol,propionic acid, propylene carbonate, propylene glycol, propylene glycolalginate, propyl gallate, propylparaben, propylparaben potassium,propylparaben sodium, protamine sulfate, rapeseed oil, Ringer'ssolution, saccharin, saccharin ammonium, saccharin calcium, saccharinsodium, safflower oil, saponite, serum proteins, sesame oil, colloidalsilica, colloidal silicon dioxide, sodium alginate, sodium ascorbate,sodium benzoate, sodium bicarbonate, sodium bisulfite, sodium chloride,anhydrous sodium citrate, sodium citrate dehydrate, sodium chloride,sodium cyclamate, sodium edentate, sodium dodecyl sulfate, sodium laurylsulfate, sodium metabisulfite, sodium phosphate, dibasic, sodiumphosphate, monobasic, sodium phosphate, tribasic, anhydrous sodiumpropionate, sodium propionate, sodium sorbate, sodium starch glycolate,sodium stearyl fumarate, sodium sulfite, sorbic acid, sorbitan esters(sorbitan fatty esters), sorbitol, sorbitol solution 70%, soybean oil,spermaceti wax, starch, corn starch, potato starch, pregelatinizedstarch, sterilizable maize starch, stearic acid, purified stearic acid,stearyl alcohol, sucrose, sugars, compressible sugar, confectioner'ssugar, sugar spheres, invert sugar, Sugartab, Sunset Yellow FCF,synthetic paraffin, talc, tartaric acid, tartrazine, tetrafluoroethane(HFC), theobroma oil, thimerosal, titanium dioxide, alpha tocopherol,tocopheryl acetate, alpha tocopheryl acid succinate, beta-tocopherol,delta-tocopherol, gamma-tocopherol, tragacanth, triacetin, tributylcitrate, triethanolamine, triethyl citrate, trimethyl-β-cyclodextrin,trimethyltetradecylammonium bromide, tris buffer, trisodium edentate,vanillin, type I hydrogenated vegetable oil, water, soft water, hardwater, carbon dioxide-free water, pyrogen-free water, water forinjection, sterile water for inhalation, sterile water for injection,sterile water for irrigation, waxes, anionic emulsifying wax, carnaubawax, cationic emulsifying wax, cetyl ester wax, microcrystalline wax,nonionic emulsifying wax, suppository wax, white wax, yellow wax, whitepetrolatum, wool fat, xanthan gum, xylitol, zein, zinc propionate, zincsalts, zinc stearate, or any excipient in the Handbook of PharmaceuticalExcipients, Third Edition, A. H. Kibbe (Pharmaceutical Press, London, UK, 2000), which is incorporated by reference in its entirety.Remington's Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin(Mack Publishing Co., Easton, Pa., 1980), which is incorporated byreference in its entirety, discloses various components used informulating pharmaceutically acceptable compositions and knowntechniques for the preparation thereof. Except insofar as anyconventional agent is incompatible with the pharmaceutical compositions,its use in pharmaceutical compositions is contemplated. Supplementaryactive ingredients also can be incorporated into the compositions.

The pharmaceutical compositions may be formulated to achieve aphysiologically compatible pH. In some embodiments, the pH of thepharmaceutical composition may be at least 5, at least 5.5, at least 6,at least 6.5, at least 7, at least 7.5, at least 8, at least 8.5, atleast 9, at least 9.5, at least 10, or at least 10.5 up to and includingpH 11, depending on the formulation and route of administration. Incertain embodiments, the pharmaceutical compositions may comprisebuffering agents to achieve a physiological compatible pH. The bufferingagents may include any compounds capabale of buffering at the desired pHsuch as, for example, phosphate buffers (e.g., PBS), triethanolamine,Tris, bicine, TAPS, tricine, HEPES, TES, MOPS, PIPES, cacodylate, MES,and others.

Routes of Administration

With regard to the invention, the IRES inhibitor, pharmaceuticalcomposition comprising the same, may be administered to the subject viaany suitable route of administration. The following discussion on routesof administration is merely provided to illustrate exemplary embodimentsand should not be construed as limiting the scope in any way.

Formulations suitable for oral administration can consist of (a) liquidsolutions, such as an effective amount of the IRES inhibitor of thepresent invention dissolved in diluents, such as water, saline, ororange juice; (b) capsules, sachets, tablets, lozenges, and troches,each containing a predetermined amount of the active ingredient, assolids or granules; (c) powders; (d) suspensions in an appropriateliquid; and (e) suitable emulsions. Liquid formulations may includediluents, such as water and alcohols, for example, ethanol, benzylalcohol, and the polyethylene alcohols, either with or without theaddition of a pharmaceutically acceptable surfactant. Capsule forms canbe of the ordinary hard- or soft-shelled gelatin type containing, forexample, surfactants, lubricants, and inert fillers, such as lactose,sucrose, calcium phosphate, and corn starch. Tablet forms can includeone or more of lactose, sucrose, mannitol, corn starch, potato starch,alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum,colloidal silicon dioxide, croscarmellose sodium, talc, magnesiumstearate, calcium stearate, zinc stearate, stearic acid, and otherexcipients, colorants, diluents, buffering agents, disintegratingagents, moistening agents, preservatives, flavoring agents, and otherpharmacologically compatible excipients. Lozenge forms can comprise theIRES inhibitor of the present invention in a flavor, usually sucrose andacacia or tragacanth, as well as pastilles comprising the IRES inhibitorof the present invention in an inert base, such as gelatin and glycerin,or sucrose and acacia, emulsions, gels, and the like containing, inaddition to, such excipients as are known in the art.

The IRES inhibitor s of the present invention, alone or in combinationwith other suitable components, can be delivered via pulmonaryadministration and can be made into aerosol formulations to beadministered via inhalation. These aerosol formulations can be placedinto pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen, and the like. They also maybe formulated as pharmaceuticals for non-pressured preparations, such asin a nebulizer or an atomizer. Such spray formulations also may be usedto spray mucosa. In some embodiments, the IRES inhibitor is formulatedinto a powder blend or into microparticles or nanoparticles. Suitablepulmonary formulations are known in the art. See, e.g., Qian et al., IntJ Pharm 366: 218-220 (2009); Adjei and Garren, Pharmaceutical Research,7(6): 565-569 (1990); Kawashima et al., J Controlled Release 62(1-2):279-287 (1999); Liu et al., Pharm Res 10(2): 228-232 (1993);International Patent Application Publication Nos. WO 2007/133747 and WO2007/141411.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The term, “parenteral” means not through the alimentary canal but bysome other route such as subcutaneous, intramuscular, intraspinal, orintravenous. The IRES inhibitor of the present invention can beadministered with a physiologically acceptable diluent in apharmaceutical carrier, such as a sterile liquid or mixture of liquids,including water, saline, aqueous dextrose and related sugar solutions,an alcohol, such as ethanol or hexadecyl alcohol, a glycol, such aspropylene glycol or polyethylene glycol, dimethylsulfoxide, glycerol,ketals such as 2,2-dimethyl-153-dioxolane-4-methanol, ethers,poly(ethyleneglycol) 400, oils, fatty acids, fatty acid esters orglycerides, or acetylated fatty acid glycerides with or without theaddition of a pharmaceutically acceptable surfactant, such as a soap ora detergent, suspending agent, such as pectin, carbomers,methylcellulose, hydroxypropylmethylcellulose, orcarboxymethylcellulose, or emulsifying agents and other pharmaceuticaladjuvants.

Oils, which can be used in parenteral formulations include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includeoleic acid, stearic acid, and isostearic acid. Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters.

The parenteral formulations in some embodiments contain from about 0.5%to about 25% by weight of the IRES inhibitor of the present invention insolution. Preservatives and buffers may be used. In order to minimize oreliminate irritation at the site of injection, such compositions maycontain one or more nonionic surfactants having a hydrophile-lipophilebalance (HLB) of from about 12 to about 17. The quantity of surfactantin such formulations will typically range from about 5% to about 15% byweight. Suitable surfactants include polyethylene glycol sorbitan fattyacid esters, such as sorbitan monooleate and the high molecular weightadducts of ethylene oxide with a hydrophobic base, formed by thecondensation of propylene oxide with propylene glycol. The parenteralformulations in some aspects are presented in unit-dose or multi-dosesealed containers, such as ampoules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions in some aspects are prepared from sterile powders, granules,and tablets of the kind previously described.

Injectable formulations are in accordance with the invention. Therequirements for effective pharmaceutical carriers for injectablecompositions are well-known to those of ordinary skill in the art (see,e.g., Pharmaceutics and Pharmacy Practice, J. B. Lippincott Company,Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), andASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630(1986)).

Additionally, the IRES inhibitors of the invention can be made intosuppositories for rectal administration by mixing with a variety ofbases, such as emulsifying bases or water-soluble bases. Formulationssuitable for vaginal administration can be presented as pessaries,tampons, creams, gels, pastes, foams, or spray formulas containing, inaddition to the active ingredient, such carriers as are known in the artto be appropriate.

It will be appreciated by one of skill in the art that, in addition tothe above-described pharmaceutical compositions, the IRES inhibitor ofthe invention can be formulated as inclusion complexes, such ascyclodextrin inclusion complexes, or liposomes.

In exemplary aspects, wherein the IRES inhibitor is an antisensemolecule, the antisense molecule may be formulated into a viral vectoror a nonviral vector, either of which are suitable for delivery intohumans. Li et al., Cancer Gene Ther 8:555-565 (2001) describes methodsof targeted gene therapy by non viral vectors. Chen et al., Mol Therdescribes gene therapy with a lentiviral gene delivery construct. Zhouet al., J Healthc Eng 4(2): 223-254 (2013) and Chen et al., CardiovascUltrasound 11:11 (2013) describes gene deliver with ultrasound. See,e.g., Chistiakov et al., Drug Deliv 19(8): 392-405 (2012) and Juliano etal., J Drug Target 21(1): 27-43(2013) and Southwell et al., Trends MolMed 18(11): 634-643 (2012) and International Patent ApplicationPublication Nos. WO2005/072703, WO1994/023699; WO2010/085665, andWO1998/018811. Examples 8 and 9 describe delivery of an exemplary miRNAthrough delivery of an adenoviral vector.

Dosages

For purposes herein, the amount or dose of the IRES inhibitoradministered should be sufficient to effect, e.g., a therapeutic orprophylactic response, in the subject or animal over a reasonable timeframe. For example, the dose of the IRES inhibitor of the presentinvention should be sufficient to treat SCA6 as described herein in aperiod of from about 1 to 4 minutes, 1 to 4 hours or 1 to 4 weeks orlonger, e.g., 5 to 20 or more weeks, from the time of administration. Incertain embodiments, the time period could be even longer. The dose willbe determined by the efficacy of the particular IRES inhibitor and thecondition of the animal (e.g., human), as well as the body weight of theanimal (e.g., human) to be treated.

Many assays for determining an administered dose are known in the art.For purposes herein, an assay, which comprises comparing the extent towhich SCA6 is treated upon administration of a given dose of the IRESinhibitor of the present invention to a mammal among a set of mammals,each set of which is given a different dose of the IRES inhibitor, couldbe used to determine a starting dose to be administered to a mammal. Theextent to which SCA6 is treated upon administration of a certain dosecan be represented by, for example, the extent to which the expressionof α1ACT is reduced. Methods of measuring protein expression are knownin the art, including, for instance, the methods described in theEXAMPLES set forth below.

The dose of the IRES inhibitor of the present invention also will bedetermined by the existence, nature and extent of any adverse sideeffects that might accompany the administration of a particular IRESinhibitor of the present invention. Typically, the attending physicianwill decide the dosage of the IRES inhibitor of the present inventionwith which to treat each individual patient, taking into consideration avariety of factors, such as age, body weight, general health, diet, sex,IRES inhibitor of the present invention to be administered, route ofadministration, and the severity of the condition being treated. By wayof example and not intending to limit the scope of the claimed subjectmatter, the dose of the IRES inhibitor of the present invention can beabout 0.0001 to about 1 g/kg body weight of the subject beingtreated/day, from about 0.0001 to about 0.001 g/kg body weight/day, orabout 0.01 mg to about 1 g/kg body weight/day.

Controlled Release Formulations

In some embodiments, the IRES inhibitors described herein can bemodified into a depot form, such that the manner in which the IRESinhibitor is released into the body to which it is administered iscontrolled with respect to time and location within the body (see, forexample, U.S. Pat. No. 4,450,150). Depot forms of IRES inhibitors of theinvention can be, for example, an implantable composition comprising theIRES inhibitors and a porous or non-porous material, such as a polymer,wherein the IRES inhibitor is encapsulated by or diffused throughout thematerial and/or degradation of the non-porous material. The depot isthen implanted into the desired location within the body of the subjectand the IRES inhibitor is released from the implant at a predeterminedrate.

The pharmaceutical composition comprising the IRES inhibitor in certainaspects is modified to have any type of in vivo release profile. In someaspects, the pharmaceutical composition is an immediate release,controlled release, sustained release, extended release, delayedrelease, or bi-phasic release formulation. Methods of formulating smallmolecular weight compounds, peptides, and oligonucleotides or nucleicacid analogs, for controlled release are known in the art. See, forexample, Qian et al., J Pharm 374: 46-52 (2009) and International PatentApplication Publication Nos. WO 2008/130158, WO2004/033036;WO2000/032218; and WO 1999/040942.

The instant compositions may further comprise, for example, micelles orliposomes, or some other encapsulated form, or may be administered in anextended release form to provide a prolonged storage and/or deliveryeffect.

Timing of Administration

The disclosed pharmaceutical compositions and formulations may beadministered according to any regimen including, for example, daily (1time per day, 2 times per day, 3 times per day, 4 times per day, 5 timesper day, 6 times per day), every two days, every three days, every fourdays, every five days, every six days, weekly, bi-weekly, every threeweeks, monthly, or bi-monthly. Timing, like dosing can be fine-tunedbased on dose-response studies, efficacy, and toxicity data, andinitially gauged based on timing used for other antibody therapeutics.

Combinations

In some embodiments, the IRES inhibitors described herein areadministered alone, and in alternative embodiments, the IRES inhibitorsdescribed herein are administered in combination with anothertherapeutic agent, e.g., another IRES inhibitor of the invention ofdifferent type (e.g., structure) or a completely different therapeuticagent.

In exemplary aspects, the IRES inhibitors described herein areadministered in combination with a compound which targets one of thefollowing genes: Bcat1, Bdkrb2, Bmp3, Ces1a, Chst1, Crabp1, Dapk1, Dbh,Foxq1, Gabbr2, Gfra1, Gnai1, Hsd11b1, IL17re, Iqsec3, Mb21d2, Mmp13,Nnat, Npy1r, Ntn1, Pmp22, Podx1, Prkcdbp, Ramp1, Rbp4, Reep1, Rgs10,S100a4, S100a5, S100a6, Sema3b, Sgce, Sh3bgr, Smoc2, Tbc1d9, Ugt8,Vldlr. The mRNA encoded by each of these genes are provided in theSequence Listing as SEQ ID NOs: 64-137. In exemplary aspects, the IRESinhibitors described herein are administered in combination with acompound which targets a gene product encoded by one of these genes.

In alternative or additional aspects, the IRES inhibitors describedherein are administered in combination with a compound which targets oneof the following genes, which are related to neuron differentiation:Vgf, Ntn1, S1c11a, Lhx2, Nrp1, Mab2, Ret, Etv4, Btg2, Cspg5, Sptbn4,Rab3a, Lamb2, Celsr3, Ntrk1, Mapk8ip3, Cln8, Nnat, Bmp7, Brsk1, Sema5a,Plxna3, Fgfr1. In exemplary aspects, the IRES inhibitors describedherein are administered in combination with a compound which targets agene product encoded by one of these genes.

In alternative or additional aspects, the IRES inhibitors describedherein are administered in combination with a compound which targets oneof the following genes, which are related to the cell cycle: Suv39h2,Ndc80, Rad51, Trip13, Cenpf, Ccnb1, SMC4, SMC2, Kif11, Cep55, Kif18a,Dlgap5, Lzts2, Katna1, Mih1, Mns1, Rps6. In exemplary aspects, the IRESinhibitors described herein are administered in combination with acompound that targets a gene product encoded by one of these genes.

In addition to the inventive methods comprising administering an IRESinhibitor, provided herein are alternative methods of treating SCA6 in asubject, comprising the step of administering to the subject (I) acompound which targets one or more genes other than the CACNA1A gene,wherein the one or more genes is selected from the group consisting of:Bcat1, Bdkrb2, Bmp3, Ces1a, Chst1, Crabp1, Dapk1, Dbh, Foxq1, Gabbr2,Gfra1, Gnai1, Hsd11b1, IL17re, Iqsec3, Mb21d2, Mmp13, Nnat, Npy1r, Ntn1,Pmp22, Podx1, Prkcdbp, Ramp1, Rbp4, Reep1, Rgs10, S100a4, S100a5,S100a6, Sema3b, Sgce, Sh3bgr, Smoc2, Tbc1d9, Ugt8, Vldlr, Vgf, Ntn1,Slc11a, Lhx2, Nrp1, Mab2, Ret, Etv4, Btg2, Cspg5, Sptbn4, Rab3a, Lamb2,Celsr3, Ntrk1, Mapk8ip3, Cln8, Nnat, Bmp7, Brsk1, Sema5a, Plxna3, Fgfr1,Suv39h2, Ndc80, Rad51, Trip13, Cenpf, Ccnb1, SMC4, SMC2, Kif11, Cep55,Kif18a, Dlgap5, Lzts2, Katna1, Mih1, Mns1, and Rps6, or (II) a compoundwhich targets a gene product encoded by said gene.

Kits

In some embodiments, the IRES inhibitor is provided as part of a kit orpackage or unit dose. “Unit dose” is a discrete amount of a therapeuticcomposition dispersed in a suitable carrier. Accordingly, providedherein are kits comprising an IRES inhibitor of the invention. Inexemplary aspects, the kit comprises an antisense molecule as describedherein.

In some embodiments, the components of the kit/unit dose are packagedwith instructions for administration to a subject. In some embodiments,the kit comprises one or more devices for administration to a subject,e.g., a needle and syringe, a dropper, a measuring spoon or cup or likedevice, an inhaler, and the like. In some aspects, the IRES inhibitor ispre-packaged in a ready to use form, e.g., a syringe, an intravenousbag, an inhaler, a tablet, capsule, etc. In some aspects, the kitfurther comprises other therapeutic or diagnostic agents orpharmaceutically acceptable carriers (e.g., solvents, buffers, diluents,etc.), including any of those described herein. In particular aspects,the kit comprises an IRES inhibitor of the invention along with thecurrent SCA6 standard of care.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

EXAMPLES Example 1

This example demonstrates an miRNA-mediated therapy for SCA6 whichprovides a selective translational block of the CACNA1A second cistron.

Spinocerebellar ataxia type 6 (SCA6) is a dominantly-inheritedneurodegenerative disease characterized by progressive ataxia andPurkinje cell degeneration and caused by abnormal expansions of thepolyglutamine (polyQ) tract in CACNA1A. Recently, it was discovered thatSCA6 is attributable to the expression of a polyQ repeat expansionwithin a second CACNA1A gene product, α1ACT and α1ACT expression isunder the control of internal ribosomal entry site (IRES) within theCACNA1A gene coding region. Here it is shown that miR-3191-5p bound toArgonaute protein-4 preferentially inhibits the translational initiationof α1ACT by eukaryotic initiation factors, eIF4AII and eIF4GII, whichdirectly act on CACNA1A IRES and enhance α1ACT translation. Furthermore,adeno-associated viral delivery of miR-3191-5p ameliorates the Purkinjecell degeneration and ataxia caused by SCA6-associated α1ACT in ourmouse model. These results suggest that miRNA-mediated selectivetranslational block of second cistron could be potential therapeutictargets in diseases caused by IRES-driven pathogenic gene products.

Presently there are no definitive treatments for neurodegenerativediseases which, based on the diversity of mechanisms, may requireprecision medicine. Spinocerebellar ataxia type 6 (SCA6) is one of themost common forms of autosomal dominant SCA, representing 10-20% ofpatients with dominantly-inherited ataxia and approximately 5/100,000(1-3). Patients with SCA6 develop slowly progressive cerebellar ataxiausually beginning at age 40-50 years associated with extensive selectivePurkinje cell degeneration (4, 5). The mutational mechanism responsiblefor SCA6 is an expanded polyglutamine (polyQ)-encoding CAG repeat in thegene, CACNA1A, thought originally only to encode the α1A (Cav2.1,P/Q-type) voltage-gated Ca2+ channel subunit (2, 6). Attempts toimplicate a disturbance of P/Q channel kinetics in SCA6 have beenunsuccessful (7, 8). It has been discovered that the CACNA1A gene, is abicistronic cellular gene, i.e., it encodes two structurally unrelatedproteins, with distinct functions, that are separately encoded withinthe same transcript (9). CACNA1A encodes both α1A subunit and a newlyrecognized transcription factor, α1ACT, within an overlapping openreading frame (ORF) of the same mRNA. This is achieved by the presenceof a novel internal ribosomal entry site (IRES) upstream of a second ORFencoding α1ACT. α1ACT bearing the polyQ expansion (α1ACT_(SCA6)) impairsits cellular and molecular functions, and α1ACT_(SCA6) causes increasedcell death in cultured cell models and cerebellar atrophy anddysfunction in a transgenic mouse model (9). As a potential therapy,ablation or elimination of complete CACNA1A expression would be lethal(9, 10), although selective elimination of α1ACT protein could be aviable strategy. MicroRNAs (MiRNAs) have been increasingly recognized toplay a role in the regulation of gene expression, in many cases by bothtranslational repression and mRNA destabilization (11, 12), but to datehave not been used to preferentially regulate the translation of diseasegenes driven by cellular IRES. Although prevailing evidence has pointedto the role of natural miRNAs in gene silencing and translationalrepression by binding to 3′ untranslated region (UTR) (or rarely 5′ UTR)of targeted mRNAs (11-16), predicted miRNA binding sites are foundthroughout the genome including both coding and non coding regions (17,18).

The CentroidFold program (19) was used to predict that CACNA1A IRES hasa stem-loop structure that plays an important role in IRES-driventranslation of α1ACT (9). The miRNA_Targets program (17) demonstratesthat miRNAs, miR-711 (Accession number_MIMAT0012734), -3191-5p(Accession number_MIMAT0022732), and -4786-3p (Accessionnumber_MIMAT0019955) bind to sequences within the stem-loop structure ofCACNA1A IRES (FIG. 5A to FIG. C). To examine the effects of these miRNAson CACNA1A IRES-driven translation, miRNAs with either a bicistronicIRES reporter vector or control vector were co-transfected into HEK293cells (FIG. 6). Dual luciferase assays revealed that miR-711, -3191-5p,and -4786-3p significantly down-regulated the CACNA1A IRES-drivenluciferase activities compared to a miRNA negative control (FIG. 1A).The effects of these miRNAs on CACNA1A-encoded C-terminal FLAG-taggedpeptides (α1A-FLAG and α1ACT-FLAG) were tested with the pathologicalpolyQ tract (Q33) in transfected HEK293 cells. Although miR-711 and-4′786-3p decreased both α1A-FLAG and α1ACT-FLAG expression, miR-3191-5pdown-regulated α1ACT-FLAG expression, but spared α1A-FLAG expression(FIG. 1B). This was the case with α1A bearing both normal (Q11) and SCA6(Q33) alleles (FIG. 7A). Quantitative real time-PCR (qRT-PCR) studies oftotal RNA showed that miR-711 and -4786-3p significantly decreasedCACNA1A mRNA (α1A mRNA) levels relative to a miRNA negative control, butthat miR-3191-5p did not affect α1A mRNA levels (FIG. 1C, FIG. 7B). Todetermine whether miR-3191-5p interacts with regions within the α1A mRNAother than the CACNA1A IRES, mutated CACNA1A IRES templates, resistantto the binding of miR-3191-5p (CACNA1A IRESmut: FIG. 1D, FIG. 8A andFIG. 8B) were prepared. CACNA1A IRESmut functioned normally both in theIRES dual luciferase assay and in α1A-FLAG and α1ACT-FLAG expressingHEK293 cells, but the inhibition by miR-3191-5p was prevented (FIG. 1Eand FIG. 1F). Finally, miR-3191-5p did not affect the expression levelsof endogenous α1A mRNA harboring the native 3′UTR sequence in HEK293cells (FIG. 1G). These findings demonstrated that miR-3191-5p directlyinteracted with CACNA1A IRES and inhibited IRES-driven translation ofα1ACT while sparing α1A expression.

miRNAs generally act on targeted mRNA in collaboration with themiRNA-induced silencing complex (miRISC)-guided Argonaute (Ago) proteins(20-24), although detailed mechanisms of the role of the four identifiedsubtypes in translational repression remain to be determined. To examinethe role of Ago proteins in the inhibitory effects of miR-3191-5p,miR-3191-5p were co-transfected with small interfering RNAs (siRNAs)targeting Ago1-4 and either a bicistronic IRES reporter vector orcontrol vector into HEK293 cells. Dual luciferase assay revealed thatthe knockdown of Ago4, but not Ago1-3, blocked the silencing effects ofmiR-3191-5p on the CACNA1A IRES-driven luciferase activities (FIG. 1H).Western blot analysis showed that the silencing of Ago4, but not Ago1-3,also prevented the down-regulation of α1ACT-FLAG expression bymiR-3191-5p (FIG. 1I). RNA immunoprecipitation (IP)-coupled qRT-PCRrevealed the binding affinities of Ago4 to both miR-3191-5p and CACNA1AIRES (FIG. 1J and FIG. 1K, FIG. 9A and FIG. 9B). While previous studieshave mainly focused on the interaction of Ago1 and Ago2 with miRNAs onthe 3′ UTR of targeted mRNA, human Ago4 has been reported to becatalytically inactive (22-24).

Both eukaryotic initiation factors (eIFs), eIF4A and eIF4G, have beenpreviously shown to be involved in the IRES-dependent translation inseveral types of virus IRESs (12), but have not been studied well in thecontext of mammalian IRES and regulation by miRNA. The effects ofeIF4AI, eIF4AII, eIF4GI, and eIF4GII on CACNA1A IRES-driven translationwere examined using our dual luciferase reporter assay. Over-expressionof either eIF4AII or eIF4GII, but not eIF4AI and eIF4GI increased theCACNA1A IRES-driven luciferase activities. The effects of eIF4AII andeIF4GII were blocked by co-transfection with miR-3191-5p (FIG. 2A).Similarly, using the vector expressing α1A-FLAG and α1ACT-FLAG, it wasfound that over-expression of either eIF4AII or eIF4GII increased bothα1A-FLAG and α1ACT-FLAG expression by western blot analysis, and thatmiR-3191-5p reversed the up-regulating effects of eIF4AII and eIF4GII onα1ACT-FLAG expression without affecting the eIF4AII and eIF4GIIexpression, respectively (FIG. 2B). Over-expression of either eIF4AII oreIF4GII did not affect the expression levels of α1A mRNA in qRT-PCRassays (FIG. 2C). The effects of miR-3191-5p depended on theinteractions with CACNA1A IRES, because when the CACNA1A IRESrnutvectors that were resistant to miR-3191-5p binding were used,miR-3191-5p did not block the up-regulating effects of eIF4AII andeIF4GII on CACNA1A IRES-driven luciferase activities (FIG. 2D) andα1ACT-FLAG expression (FIG. 2E). It was also found that eIF4AIIassociated with eIF4GII (FIG. 2F), which was consistent with the resultsof recent reports (25, 26). Dual luciferase assay revealed that eIF4GII,rather than eIF4AII, played a critical role in the initiation of theCACNA1A IRES-driven translation (FIG. 2G). Finally, the bindingaffinities of both eIF4AII and eIF4GII to CACNA1A IRES were tested usingtruncated transgenes of CACNA1A second cistron that lacked the sequenceof 5′ up-stream from CACNA1A IRES (IRESstopmut-α1ACT: FIG. 10A and FIG.10B), because both eIF4AII and eIF4GII affected not only CACNA1A-IRESdriven translation of α1ACT but also cap-dependent α1A translation (FIG.2B). RNA IP-coupled qRT-PCR revealed that both eIF4AII-specific andeIF4GII-specific antibodies precipitate mRNA transcribed fromIRESstopmut-α1ACT vectors and that miR-3191-5p significantly decreasedthe binding affinities of both eIF4AII and eIF4GII to CACNA1A IRES (FIG.2H). Based on these findings, it was concluded that miR-3191-5p, incollaboration with Ago4, inhibits the initiation of IRES-driventranslation of α1ACT by the complex of eIF4AII and eIF4GII that directlyacts on CACNA1A IRES to enhance IRES-driven translation.

It has already characterized the independent physiological and cellularpathological properties of wild type (WT) α1ACT and α1ACT_(SCA6) (9) invivo although these transgenes lacked the CACNA1A IRES and produced arather mild phenotype. Elimination of the portion of CACNA1A IRESsequence from the human mRNA encoding α1A-Q33 selectively eliminates theexpression of α1ACT fragment and is protective in a cell culture modelof α1ACT_(SCA6) toxicity without affecting α1A expression (9). It wasreasoned that targeted expression of α1ACT, in which translation ofα1ACT is under the control of CACNA1A IRES, would more accuratelyresemble SCA6 pathogenesis in a mouse model, while providing a suitabletarget for IRES-directed miRNA based therapy. Thus, an in vivoadeno-associated viral (AAV) delivery system was developed using theconstructs of α1ACT transgene tagged with a C-terminal FLAG epitope,expressed under the control of CACNA1A IRES (AAV-α1ACT-Q11, or -Q33)(FIG. 11A). The control AAV vector expressing Aequorea coerulescensgreen fluorescent protein (GFP) (AAV-GFP) was also prepared. A viralload of 10⁹ vector genomes (vg) of each of these constructs was directlyinjected into the right lateral ventricle of neonatal WT mice ofC57/BL6J at post-natal day 1. Four weeks after AAV vector injection,AAV-injected WT mice were sacrificed and a widespread transduction ofthe viral particles was found throughout the mouse brain and cerebellum.Western blot analysis revealed the strong expression of GFP,α1ACT-Q11-FLAG, or α1ACT-Q33-FLAG in the brain and cerebellum of WT miceinjected with AAV-GFP (WT-GFP mice), AAV-α1ACT-Q11 (WT-α1ACT-Q11 mice),or AAV-α1ACT-Q33 (WT-α1ACT-Q33 mice) (FIG. 11B). The persistentexpression of GFP, α1ACT-Q11-FLAG, or α1ACT-Q33-FLAG was confirmed forat least 12 weeks of our observation period (FIG. 11B). qRT-PCR studiesof total RNA from the cerebellum of WT-α1ACT-Q11 mice and WT-α1ACT-Q33mice showed that the expression levels of mRNA transcribed from thetransgenes delivered by AAV were comparable (FIG. 11C). The presence ofthe AAV vector in the Purkinje cells of AAV-injected WT mice wasvisually confirmed by the co-localization of GFP or FLAG and stainingfor calbindin, a Purkinje cell marker (FIG. 3A). As compared to WT-GFPmice and WT-α1ACT-Q11 mice, WT-α1ACT-Q33 mice showed significantthinning of the molecular layer, decrease in the density of dendritictree and number of Purkinje cells in the cerebellum (FIG. 3B to FIG.3E). Although WT-α1ACT-Q33 mice grew at roughly the same body weight asWT-GFP mice and WT-α1ACT-Q11 mice (FIG. 3F), WT-α1ACT-Q33 mice showedsignificant defects in motor functions assessed by rotarod (FIG. 3G),activities by open field assay (FIG. 3H), and gait stability by avideo-assisted computerized treadmill (FIG. 12A to FIG. 12F). TheDigigait assay revealed that WT-α1ACT-Q33 mice exhibited shorter stridelength (FIG. 12A, FIG. 12C, and FIG. 12E) and greater stride frequencies(FIG. 12B, FIG. 12D, and FIG. 12F) from 4 weeks old, indicatingprogressive instability during walking. These findings indicated thatviral administration and expression of α1ACT_(SCA6) under the control ofCACNA1A IRES resembled the pathological and clinical features of SCA6(3, 4).

Next, to address the therapeutic effects of the reduction of IRES-driventranslation of α1ACT_(SCA6) by miR-3191-5p on our SCA6 mouse model, weconstructed an AAV vector that allowed for the simultaneous expressionof GFP and either miR-3191-5p (AAV-miR-3191-5p) or a nonspecific miRNA,miR-mock (AAV-miR-mock). We then co-injected AAV-α1ACT-Q33 with eitherAAV-miR-3191-5p or AAV-miR-mock into the right lateral ventricle ofneonatal WT mice of C57/BL6J at post-natal day 1. Western blot analysisrevealed the persistent and widespread expression of α1ACT-Q33-FLAG orGFP in the brain and cerebellum of mice co-injected with AAV-α1ACT-Q33and either AAV-miR-mock (WT-Q33-miR-mock mice) or AAV-miR-3191-5p(WT-Q33-miR-3191-5p mice) for at least 12 weeks and the significantlydecreased α1ACT-Q33-FLAG protein levels in the brain and cerebellum ofWT-Q33-miR-3191-5p mice as compared to WT-Q33-miR-mock mice (FIG. 13).WT-Q33-miR-3191-5p mice showed the same expression levels of mRNAtranscribed from the transgenes delivered by AAV-α1ACT-Q33 in thecerebellum as compared to WT-Q33-miR-mock mice (FIG. 13).Immunohistochemical examination of mouse cerebellum showed normalthickness of the molecular layer, density of dendritic tree and numberof Purkinje cells in the cerebellum of WT-Q33-miR-3191-5p mice (FIG. 4Ato FIG. 4D) compared with WT-Q33-miR-mock mice. Although the treatmentwith AAV-miR-3191-5p did not affect the clinical symptoms of body weightin WT-Q33-miR-3191-5p mice compared to WT-Q33-miR-mock mice (FIG. 4E),we found that the α1ACT_(SCA6)-associated disease phenotypes inWT-Q33-miR-3191-5p mice were prevented, assessed by rotarod (FIG. 4F),open field assay (FIG. 4G), and a video-assisted computerized treadmillfor gait analysis (FIG. 14A to FIG. 14F). We also monitored WT miceinjected with AAV-miR-3191-5p at postnatal day 1 to examine the adverseoff-target effects of the treatment with AAV-miR-3191-5p. We found noobvious adverse effects in mouse behavior or morphological anomalies inthe brain, cerebellum, heart, lung, liver, and kidney of the WT miceinjected with AAV-miR-3191-5p for at least 12 weeks (FIG. 15).

In summary, we discovered that miR-3191-5p directly acts on the CACNA1AIRES to regulate the translation of α1ACT, which requires the presenceof Ago4. We have also demonstrated that miR-3191-5p bound to Ago4preferentially inhibits the translational initiation of α1ACT byeukaryotic initiation factors, eIF4AII and eIF4GII, which enhance α1ACTtranslation. We further showed that the inhibition of the CACNA1AIRES-driven α1ACT_(SCA6) expression by the AAV vector-mediated deliveryof miR-3191-5p has a therapeutic effect on the α1ACT_(SCA6)-induced SCA6phenotypes in our model mouse.

Our results are the first to point to a specific role of a natural miRNAin repressing translation by the direct interaction with a site withincellular IRES (16, 27, 28). There are four members of the RNA-inducedsilencing complex-related Ago protein family in human, Ago1, Ago2, Ago3,and Ago4 (22). Very little is known about Ago4, although the absence ofa catalytic domain suggests that it might have a distinct role in genesilencing compared with Ago2 (29-34). Our findings that Ago4 appears toselectively interact with a miRNA and the CACNA1A IRES to represstranslation may suggest it has a specific role for mRNA-sparingtranslational repression via the miRISC, possibly even selectively fortranslational repression of IRESs. Finally, because therapy targetingIRES activity has been a mainstay of anti-viral therapy and not beenemployed for genetic disease (35-37), this work will prepare us for thedevelopment of therapies using a novel strategy by the selectivesuppression of IRES-driven pathogenic gene products based on delivery ofdisease-specific miRNAs in future.

This example demonstrates a novel miRNA-mediated therapeutic approachfor spinocerebellar ataxia type 6 via the selective translational blockof second cistron in CACNA1A gene.

Example 2

This example provides a description of the materials and methods used inthe experiments of Example 8.

Construction of DNA Plasmids

We used pcDNA3 vectors expressing CACNA1A-encoded FLAG-tagged peptides(α1A-FLAG and α1ACT-FLAG) and a bicistronic IRES reporter vector orcontrol vector as described previously (9).

To obtain mutated transgenes that are resistant to the binding ofmiR-3191-5p (CACNA1A IRESmut vectors), we performed C>G and G>Csubstitutions within the predicted miR-3191-5p binding site. We alsoperformed C>G and G>C substitutions within the complementary bindingsite of the sequence targeted by miR-3191-5p to maintain the structureof CACNA1A IRESmut same as that of the original (FIG. 1D, FIG. 8A andFIG. 8B). Briefly, we amplified the predicted binding site ofmiR-3191-5p using primers miR mut forward:5′-ATGTCTCCGCCCCTGGGTCTccccAAcAAcTcTggccCCAGAGTGGCTTACAAGCG-3′ (SEQ IDNO: 182) and miR mut reverse:5′-CGCTTGTAAGCCACTCTGGggccAgAgTTgTTggggAGACCCAGGGGCGGAGACAT-3′ (SEQ IDNO: 183), the complementary binding site of the sequence targeted bymiR-3191-5p using primers anti-miR mut forward:5′-ACTGAGCACAATAACTTggccAggTTgTTgg AGGCCCTCATGCTTCTC-3′ (SEQ ID NO: 184)and anti-miR mut reverse:5′-GAGAAGCATGAGGGCCTccAAcAAccTggccAAGTTATTGTGCTCAGT-3′ (SEQ ID NO: 185).Then we annealed PCR products using primers anti-miR mut forward and miRmut reverse and inserted the annealed PCR products into the BIpI sitesof pcDNA3 vectors expressing CACNA1A-encoded FLAG-tagged peptides andthe EcoRI and Ncol sites of a bicistronic IRES reporter vector.

We also prepared the truncated transgenes of CACNA1A second cistron thatlacked the sequence of 5′ up-stream from CACNA1A IRES(IRESstopmut-α1ACT: FIG. 10A and FIG. 10B). Briefly, the sequence of4962 to 7757 nt of full-length CACNA1A (NM_001127222.1, NP_001120694.1),correspondent to the sequence of CACNA1A IRES and α1ACT open readingframe, was amplified using primers TRESstopmut-α1ACT forward:5′ATCAGGATCCGCCCTCAACACCATCGTGC-3′ (SEQ ID NO: 186) andIRESstopmut-α1ACT reverse: 5′-GAATCTAGATTACTTGTCATCG-3′ (SEQ ID NO: 187)from pcDNA3 vectors expressing CACNA1A-encoded FLAG-tagged peptides. ThePCR product was inserted into the BamHI and XbaI sites of pcDNA3vectors. We also modified the sequence of 5013 to 5015 nt from “TAT” to“TAG” of stop codon.

pDEST-GFP-Ago4 is a kind gift from E. Chan (Addgene plasmid #21536)(38). pcDNA3-HA-eIF4GI and pcDNA3-HA-eIF4GII are kind gifts from N.Sonenberg (McGill University, Canada) (39). M. Bushell (Medical ResearchCouncil, UK) kindly provided us with the plasmid expressing human eIF4AIand eIF4AII (13). pcDNA3.1-HisXpress vector was a kind gift from T.Cooper (Baylor College of Medicine). From the plasmids expressing humaneIF4AI and eIF4AII, we amplified human eIF4AI and eIF4AII cDNA usingprimers eIF4AI-PCR forward: 5′-ATCAGGATCCATGTCTGCGAGCCAGGAT-3′ (SEQ IDNO: 188); eIF4AI-PCR reverse: 5′-ATCAGGGCCCAGGTCAGCAACATTGAGG-3′ (SEQ IDNO: 189); and eIF4AII-PCR forward: 5′-ATCAGGATCCATGTCTGGTGGCTCCGCG-3′(SEQ ID NO: 190); eIF4AII-PCR reverse: ATCAGGGCCCTTAAATAAGGTCAGCCAC-3′(SEQ ID NO: 191), respectively. Then we inserted PCR products into BamHIand ApaI digested pcDNA3.1-HisXpress vectors to obtainpcDNA3.1-HisXpress-eIF4AI or -eIF4AII. We used the original eitherpcDNA3.1/HisC or pcDNA3-HA vectors lacking the insertions in themultiple cloning sites as control vectors.

Co-Transfection of DNA Plasmids with Either Synthetic miRNA or siRNAinto Cultured Cells

HEK293 cells were cultured in DMEM supplemented with 10% FBS. All siRNAsand miRNAs were purchased from Life Technologies. ID numbers are shownas follows: Ago1 (s25500); Ago2 (s25931); Ago3 (s46947); Ago4 (s46949);eIF4AII (s4570); eIF4GII (s16519); a siRNA negative control(#12935-300); miR-711 (MC15715); miR-3191-5p (MC23769); miR-4786-3p(MC21295); and a miRNA negative control (#4464059). We plated HEK293cells onto six-well dishes and co-transfected each dish with 1.0 μg ofthe vector expressing the following: a bicistronic IRES reporter,control reporter, and α1A-FLAG and α1ACT-FLAG; 0.5 μg of the vectorexpressing the following: eIF4AI, eIF4AII, eIF4GI, and eIF4GII; andeither 20 nM synthetic miRNA or 20 nM siRNA molecules. We usedLipofectamine 2000 (Life Technologies) as a transfection reagent in allcases. Neither the siRNA negative control nor the miRNA negative controlmatched any human mRNA. The transfected cells were cultured for 48 hrsbefore being processed for RNA and protein analysis.

Luciferase Assay

HEK293 cells were plated onto six-well plates and co-transfected with abicistronic IRES reporter vector or control vector. 48 hrs afterco-transfection, luciferase activities were determined using theDual-Luciferase Assay System (Promega) as manufacturer's instructions.The activities of Firefly and Renilla luciferase in lysates preparedfrom transfected cells using the Dual-Luciferase Assay System weremeasured by using a Wallac 1420 VICTOR 3 V luminometer with a 1 secintegration time (Perkin Elmer, Waltham, Mass.).

Protein Expression Analysis

We did western blot analysis as previously described (9, 40). We usedthe following primary antibodies: FLAG-specific antibody (1:5,000, A8592and 1:5,000, F1804; Sigma-Aldrich); Ago1-specific antibody (1:2,000,9388S; Cell Signaling Technology); Ago2-specific antibody (1:2,000,SAB4200085; Sigma-Aldrich); Ago3-specific antibody (1:2,000, 5054S; CellSignaling Technology); Ago4-specific antibody (1:2,000, 6913S; CellSignaling Technology); eIF4AII-specific antibody (1:2,000, ab31218;Abcam); eIF4GII-specific antibody (1:1,000, sc-100732; Santa CruzBiotechnology); GFP-specific antibody (1:2,000, M048-3; MBL);GAPDH-specific antibody (1:5,000, AM4300; Life Technologies).

Quantitative Real-Time PCR

The total RNA was extracted from HEK293 cells and the brain andcerebellum of mice using the miRNeasy Mini Kit (Qiagen) and reversetranscribed using the Superscript VILO (Life Technologies) for mRNA andNCode VILO (Life Technologies) for miRNA. The complementary DNAs werethen used for real time PCR using the iQ SYBR Green Supermix (Bio-RadLaboratories). We did the amplification, detection, and data analysisusing a Bio-Rad iCycler system (Bio-Rad Laboratories). The crossingthreshold values for the mRNAs of the individual genes were normalizedto beta-actin. The crossing threshold values for the miRNA of theindividual genes were normalized to the U6 small nuclear RNA. Changes inthe expression of mRNA and miRNA were expressed as a fold changerelative to the control. We used the following primers in this study.The sequences of the hsa-CACNA1A primers were: forward,5′-GTCTGGGGAAGAAGTGTCCG-3′ (SEQ ID NO: 192); reverse,5′-GCTCCTCCCTTGGCAATCTT-3′ (SEQ ID NO: 193). These hsa-CACNA1A primersdiscriminated between the human CACNA1A mRNA and the mouse CACNA1A mRNA.The sequences of the luciferase reporter primers were: forward,5′-TTTACATGGTAACGCGGCCT-3′ (SEQ ID NO: 194); reverse,5′-GGTCTGGTATAATACACCGCGC-3′ (SEQ ID NO: 195). The sequences of thehsa-beta-actin primers were: forward, 5′-GCGGGAAATCGTGCGTGACATT-3′ (SEQID NO: 196); reverse, 5′-GATGGAGTTGAAGGTAGTTTCGTG-3′ (SEQ ID NO: 197).The sequences of the mmu-beta-actin primers were: forward,5′-GCTACAGCTTCACCACCACA-3′ (SEQ ID NO: 198); reverse,5′-TCTCCAGGGAGGAAGAGGAT-3′ (SEQ ID NO: 199). The sequences of themiR-3191-5p primers were: forward, 5′-GCTCTCTGGCCGTCTAC-3′ (SEQ ID NO:200); reverse, 5′-GTCCAGTTTTTTTTTTTTTTTGGAAG-3′ (SEQ ID NO: 201). Thesequences of the U6 small nuclear RNA primers were: forward,5′-CTTCGGCAGCACATATACTAAA-3′ (SEQ ID NO: 202); reverse,5′-AAAATATGGAACGCTTCACG-3′ (SEQ ID NO: 203). We designed the miR-3191-5pprimers using a bioinformatics program (41).

Immunoprecipitation.

We plated HEK293 cells onto 100 mm dishes and co-transfected each dishwith 3.0 μg of the vector expressing eIF4AII and eIF4GII. 48 hrs aftertransfection, we harvested HEK293 cells for immunoprecipitation usingeIF4AII-specific antibody (5 μg per sample, ab31218; Abcam),eIF4GII-specific antibody (5 μg per sample, sc-100732; Santa CruzBiotechnology), and Dynabeads™ Protein G Immunoprecipitation Kit (LifeTechnologies) according to the manufacturer's suggested protocols. Anrabbit immunoglobulin G (5 μg per sample, PP64B; Millipore) and mouseimmunoglobulin G (5 μg per sample, CS200621; Millipore) were used ascontrols.

Immunoprecipitation-Coupled qRT-PCR.

We plated HEK293 cells onto 100 mm dishes and co-transfected each dishwith 2.0 μg of the vector expressing the following: full-lengthCACNA1A-Q33-encoded FLAG-tagged peptides, GFP-miR-3191-5p (SC401396;OriGene), GFP-Ago4, bicistronic IRES reporter, control reporter,eIF4AII, eIF4GII, or the truncated transgenes of CACNA1A second cistronlacking the sequence of 5′ up-stream from CACNA1A IRES(IRESstopmut-α1ACT). We used the original either pcDNA3.1/HisC orpcDNA3-HA vectors lacking the insertions in the multiple cloning sitesas control vectors. 48 hrs after transfection, we harvested HEK293 cellsfor co-immunoprecipitation using Ago4-specific antibody (5 μg persample, 6913S; Cell Signaling Technology), eIF4AII-specific antibody (5μg per sample, ab31218; Abcam), eIF4GII-specific antibody (5 μg persample, sc-100732; Santa Cruz Biotechnology), and Magna RIP™ RNA-BindingProtein Immunoprecipitation Kit (Millipore) according to themanufacturer's suggested protocols. An rabbit immunoglobulin G (5 μg persample, PP64B; Millipore) and mouse immunoglobulin G (5 μg per sample,CS200621; Millipore), supplied by the manufacturer, were used ascontrols. Immunoprecipitated RNA was converted to cDNA using SuperscriptVILO (Life Technologies) for mRNA and NCode VILO (Life Technologies) formiRNA and analyzed by qRT-PCR for the differential expression of α1A-Q33mRNA and IRESstopmut-α1ACT mRNA using the following primers: forward,5′-GTCTGGGGAAGAAGTGTCCG-3′ (SEQ ID NO: 192); reverse,5′-GCTCCTCCCTTGGCAATCTT-3′ (SEQ ID NO: 193), miR-3191-5p: forward,5′-GCTCTCTGGCCGTCTAC-3′ (SEQ ID NO: 200); reverse,5′-GTCCAGTTTTTTTTTTTTTTTGGAAG-3′ (SEQ ID NO: 201), a bicistronic IRESreporter vector: forward, 5′-TGCTGACTGTTTTCCAGTGC-3′ (SEQ ID NO: 204);reverse, 5′-AAGGAGCCGATGATGATGAG-3′ (SEQ ID NO: 205), and controlreporter vector: forward, 5′-TTTACATGGTAACGCGGCCT-3′ (SEQ ID NO: 194);reverse, 5′-GGTCTGGTATAATACACCGCGC-3′ (SEQ ID NO: 195).

Development of the Adeno-Associated Virus (AAV) Vectors Expressing FLAGTagged α1ACT Under the Control of CACNA1A IRES or SimultaneousExpression of miRNA and GFP.

The AAV vector plasmids contained an expression cassette consisting of ahuman cytomegalovirus immediate-early promoter, followed by a target DNAor miR, and a simian virus 40 polyadenylation signal sequence betweenthe inverted terminal repeats of the AAV3 genome. The AAV9 vp cDNA wassynthesized as previously described with the substitution of thymidinefor adenine 1337, which introduced an amino acid change from tyrosine tophenylalanine at position 446 (42). Recombinant AAV vectors wereproduced by transient transfection of HEK293 cells using the vectorplasmid, an AAV3 rep and AAV9 vp expression plasmid, and the adenoviralhelper plasmid pHelper (Agilent Technologies, Santa Clara, Calif.). Therecombinant viruses were purified by isolation from two sequentialcontinuous CsCl gradients, and the viral titers were determined byqRT-PCR. The viral vectors used for expression of α1ACT with normalrepeat size (AAV-α1ACT-Q11) and mutant repeat size (AAV-α1ACT-Q33)contained the truncated transgenes of CACNA1A second cistron that lackedthe sequence of 5′ up-stream from CACNA1A IRES (IRESstopmut-α1ACT) (FIG.11A). AAV-GFP contained the Aequorea coerulescens GFP. The sequences ofmiR-3191-5p and miR-mock are as follows. miR-3191-5p:

(SEQ ID NO: 181) 5′GGGGTCACCTCTCTGGCCGTCTACCTTCCACACTGACAAGGGCCGTGGGGACGTAGCTGGCCAGACAGGTGA CCCC-3′; miR-mock:(SEQ ID NO: 206) 5′-GTATTGCGTCTGTACACTCACCGTTTTGGCCACTGACTGACGGTGAGTGCAGACGCAATA-3′.

Injection of AAV into the Ventricle of Neonatal Wild-Type (WT) Mice.

C57/BL6J mice were purchased from Jackson Laboratory and maintained inour breeding colony. At post-natal day 1, neonatal mice wereindividually anesthetized on ice, and a total of 10⁹ vector genomes in3-5 μl of AAV solution was injected into the right lateral ventriclewith a Hamilton syringe 10 ul, 32G (Hamilton Company). The viralsolution contained 0.04% trypan blue (Sigma-Aldrich) to help determineif the ventricles were indeed injected. Only those neonatal WT mice inwhich the lateral ventricles were filled with viral solution wereanalyzed. 6 male and 6 female mice were enrolled into each group: twogroups of WT-GFP mice; WT-α1ACT-Q11 mice; WT-α1ACT-Q33 mice; WT-Q33-mockmice; and WT-Q33-miR-3191-5p mice. All animal experiments were approvedand carried out in accordance with the regulations and guidelines forthe care and use of experimental animals at the Institutional AnimalCare and Use Committee of the University of Chicago.

The Neurological and Behavioral Assessment of the WT Mice Injected withAAV.

We examined the neurological and behavioral assessments of all mice at4, 8, and 12 weeks of age.

Rotarod-task. We analyzed rotarod task of mice using an Economex Rotarod(Colombus Instruments, Colombus, Ohio) with accelerating mode (4-40 rpm,acceleration with 0.1 rpm per 0.8 sec). We performed three consecutivetrials and recorded the longest duration on the rod for each mouse.

Open field assay. We examined open field assay using Mouse Open FieldArena and 48 Cannel IR Controller for Open Field Activity (ENV-510 andENV-520; Med Associates, Inc., St Albans City, Vt.). Briefly, mice wereplaced in the center of open field area and their movements weremonitored through the side-mounted photobeams for 30 min. We analyzedmultiple parameters using Activity Monitor software (Med Associates,Inc., St Albans City, Vt.) and adopted total distance traveled to assessthe activity of each mouse.

Digigait analysis. We also examined a video-assisted computerizedtreadmill for mouse gait analysis using a Digigait with Digigaitsoftware (Mouse Specifics, Framingham, Mass.). All mice were tested atthe speed of 25 cm/sec. Stance length was normalized to mouse bodylength.

Immunohistochemistry, Immunofluorescence, and Histopathology

Immunohistochemistry was performed as previously reported except asmodified below (43). Briefly, paraffin-embedded sections of perfusedbrains were de-waxed and rehydrated, then steamed for 20 min in antigenretrieval solution (Reveal; Biocare Medical, Walnut Creek, Calif.).Sections were blocked and exposed to primary antibody for 12 hrs at 4°C. After washing, fluorescent secondary antibody in PBS-T (phosphatebuffered saline and 0.05% Tween-20) was added for 1 hr at roomtemperature. Confocal fluorescence images were captured with a Leica TCSSP2 laser scanning confocal microscope (Leica Microsystems, Inc.,Buffalo Grove, Ill.). We used NIH Image J software to quantify thepercentage of specific expression of antibody in each sample. Themolecular layer thickness and density of Purkinje dendritic trees werecalculated as previously described (9, 44). Purkinje cells selected forthe measurements spanned the molecular layer were well-stained and werenot obscured by adjacent cells. The dendritic trees of the capturedPurkinje cell image and the area enclosed were outlined and measuredusing NIH Image J software. 50-100 Purkinje cells were analyzed in eachsample to calculate the mean of the density of Purkinje dendritic trees.To assess the number of Purkinje cells in cerebellum, we calculated thenumber of Purkinje cells in the entire area and expressed the results asthe number per 250 μm. We used the following primary antibodies:FLAG-specific antibody (1:200, F1804; Sigma-Aldrich); GFP-specificantibody (1:200, M048-3; MBL); calbindin-specific antibody (1:200,CB38a; Swant). Goat AlexaFluor 488-conjugated anti-rabbit and goatAlexaFluor 594-conjugated anti-mouse IgG antibodies (Lifetechnologies)were used for secondary fluorescence detection. For the tissue sectionsstained with Hematoxilyn and eosin staining, digital image files werecreated with a 3D Histech Pannoramic Scan whole slide scanner (PerkinElmer, Waltham, Mass.) with a Stingray F146C color camera (Allied VisionTechnologies, Stadtroda, Germany). Individual images were created withthe 3D Histech Pannoramic Viewer software (Perkin Elmer, Waltham,Mass.).

Statistical Analysis

All data represent 3 biological repeats unless stated otherwise. Valuesrepresent mean±standard error of the mean (S.E.M.). Differences betweenexperimental groups were compared by Student's t-test (two tailed), ortwo-way ANOVA with Bonferroni post-test when multiple comparisons weremade. Statistical significance in figures: *P<0.05, **P<0.01,***P<0.001, n.s.: not significant.

Example 3

This example demonstrates selectively turning off disease genes withoutdisrupting other processes, a growing goal of genetic research. In thisexample, we work with a gene that expresses two proteins, a calciumchannel, necessary for life, and a regulatory protein, α1ACT, which,when mutated, causes a form of ataxia called SCA6. We figured out how toblock expression of the disease protein without affecting the calciumchannel using a small sequence of RNA called miRNA. We then used a viralvector to deliver this miRNA to mice engineered to develop a severe formof SCA6 and successfully prevented the disease.

Spinocerebellar ataxia type 6 (SCA6) is a dominantly inheritedneurodegenerative disease characterized by slowly progressive ataxia andPurkinje cell degeneration. SCA6 is caused by a polyglutamine repeatexpansion within a second CACNA1A gene product, α1ACT. α1ACT expressionis under the control of an internal ribosomal entry site (IRES) presentwithin the CACNA1A coding region. Whereas SCA6 allele knock-in mice showindistinguishable phenotypes from wild-type littermates, expression ofSCA6-associated α1ACT (α1ACTSCA6) driven by a Purkinje cell-specificpromoter in mice produces slowly progressive ataxia and cerebellaratrophy. We developed an early-onset SCA6 mouse model using anadeno-associated virus (AAV)-based gene delivery system to ectopicallyexpress CACNA1A IRES-driven α1ACTSCA6 to test the potential of CACNA1AIRES-targeting therapies. Mice expressing AAV9-mediated CACNA1AIRES-driven α1ACTSCA6 exhibited early-onset ataxia, motor deficits, andPurkinje cell degeneration. We identified miR-3191-5p as a microRNA(miRNA) that targeted CACNA1A IRES and preferentially inhibited theCACNA1A IRES-driven translation of α1ACT in an Argonaute 4(Ago4)-dependent manner. We found that eukaryotic initiation factors(eIFs), eIF4AII and eIF4GII, interacted with the CACNA1A IRES to enhanceα1ACT translation. Ago4-bound miR-3191-5p blocked the interaction ofeIF4AII and eIF4GII with the CACNA1A IRES, attenuating IRES-driven α1ACTtranslation. Furthermore, AAV9-mediated delivery of miR-3191-5pprotected mice from the ataxia, motor deficits, and Purkinje celldegeneration caused by CACNA1A IRES-driven α1ACTSCA6. We haveestablished proof of principle that viral delivery of an miRNA canrescue a disease phenotype through modulation of cellular IRES activityin a mouse model.

Introduction

Spinocerebellar ataxias (SCAs) are a genetically heterogeneous group ofdominantly inherited neurodegenerative diseases characterized byprogressive ataxia and Purkinje cell degeneration (1-3). To date, morethan 30 SCAs have been characterized, each being associated withdistinct genes and mutations and therefore requiring individualtherapeutic approaches (2, 3). The lack of efficacious therapeutics andthe large number of genetic events that result in SCAs have highlightedthe need for effective preclinical models to identify and test“druggable” targets.

SCA type 6 (SCA6) is one of the most common forms of autosomal dominantSCAs, representing 10 to 20% of patients with dominantly inheritedataxia. It has an incidence of about 5/100,000 persons (2-8). Patientswith SCA6 develop slowly progressive cerebellar ataxia with extensiveselective Purkinje cell degeneration, usually beginning at 40 to 50years of age (2-8). SCA6 is caused by an expanded CAG repeat in theCACNA1A gene, which results in an expanded polyglutamine (polyQ) tract.Previous studies unexpectedly found that the expanded polyQ tract doesnot affect the function or kinetics of the α1A (Cav2.1, P/Q-type)voltage-gated Ca2+ channel subunit, a gene product of the full-lengthCACNA1A gene (9, 10). Additionally, SCA6 allele knock-in mice wereindistinguishable from wild-type littermates, even in old age (9, 10).

We recently discovered that the CACNA1A gene is bicistronic, that is, itencodes both the full-length α1A subunit and a newly recognizedtranscription factor, α1ACT, consisting of 547 amino acids of the Cterminus encoded within a separate open reading frame (ORF) of the samemRNA. The second cistron is translated from a newly identified internalribosomal entry site (IRES) upstream of the second ORF. We have alsocharacterized the cellular physiological and pathological properties ofboth wild-type α1ACT and an expanded polyQ tract containing α1ACT invivo, showing that the expanded polyQ tract in α1ACT results in the SCA6phenotype. Additionally, we showed that elimination of the portion ofthe IRES sequence from the human mRNA encoding SCA6-associated α1Aselectively eliminated the expression of the SCA6-associated α1ACT(α1ACTSCA6) fragment and was protective in a cell culture model ofα1ACTSCA6 toxicity (11). Because the complete silencing of CACNA1A geneexpression would be lethal (11, 12), a more suitable therapeuticapproach for SCA6 would be to selectively eliminate expression of α1ACTwhile sparing α1A expression. We reasoned that modulating the expressionof microRNAs (miRNAs) targeting the CACNA1A IRES could offer a newtherapeutic approach for treating SCA6 through regulation ofIRES-dependent α1ACTSCA6 translation. On the basis of this hypothesis,we developed a mouse model in which α1ACTSCA6 was expressed in a CACNA1AIRES-dependent manner.

Results

Somatic Gene Transfer of CACNA1A IRES-Driven α1ACTSCA6 Causes PurkinjeCell Degeneration in Mice

We established an in vivo adeno-associated virus type 9 (AAV9) deliverysystem using the constructs of α1ACT transgenes tagged with a C-terminalFLAG epitope, expressed under the control of CACNA1A IRES(IRES-α1ACT-Q11 and IRES-α1ACT-Q33, FIG. 24A; AAV9-α1ACT-Q11 andAAV9-α1ACT-Q33, FIG. 24B). We also prepared a control AAV9 vectorexpressing green fluorescent protein (GFP) (AAV9-GFP). A viral load of1010 vector genomes (vg) of each of these constructs was directlyinjected into the right lateral ventricle of neonatal wild-type C57/BL6Jmice at postnatal day 1. In vivo delivery of AAV9 by intraventricularinjection resulted in more than 75% of Purkinje cells being transducedwith the AAV9 vector (FIG. 24C and FIG. 24D). Four weeks after AAV9vector injection, we sacrificed the mice and found widespread expressionof α1ACT-Q11-FLAG or α1ACT-Q33-FLAG that was predominantly observed inthe cerebral cortex and cerebellum of wild-type mice injected withAAV9-α1ACT-Q11 (AAV9-α1ACT-Q11 mice) or AAV9-α1ACT-Q33 (AAV9-α1ACT-Q33mice) (FIG. 16A, FIG. 16B). Quantitative real-time polymerase chainreaction (qRT-PCR) of total RNA from the cerebellum of AAV9-injectedmice revealed that α1ACT mRNA in the cerebellum of AAV9-α1ACT-Q11 andAAV9-α1ACT-Q33 mice showed a greater than 2.5-fold up-regulationcompared to mouse endogenous CACNA1A mRNA in the cerebellum of wild-typemice injected with AAV9-GFP (AAV9-GFP mice). Expression ofIRES-α1ACT-Q11 mRNA in AAV9-α1ACT-Q11 mice was comparable to that ofIRES-α1ACT-Q33 mRNA in AAV9-α1ACT-Q33 mice (FIG. 16C). CACNA1AIRES-driven α1ACT protein expression in the cerebellum was alsocomparable between AAV9-α1ACT-Q11 and AAV9-α1ACT-Q33 mice (FIG. 16D).

We found that, compared with AAV9-α1ACT-Q11 mice, AAV9-α1ACT-Q33 miceshowed a significant decrease in the molecular layer thickness (P<0.01)and the density of the Purkinje cell dendritic tree (P<0.01) (FIG.16E-FIG. 16G). We also found that AAV9-α1ACT-Q33 caused a 50% loss ofPurkinje cells in the cerebellum (FIG. 16H) but no obvious pathologicalchanges in the cerebral cortex and hippocampus (FIG. 25A and FIG. 25B).These pathological features in the cerebellum of AAV9-α1ACT-Q33 miceresembled those of Purkinje cell degeneration in patients with SCA6(13-15).

CACNA1A IRES-Driven α1ACTSCA6 Causes an Early-Onset Ataxia and MotorDeficits in Mice

To assess the behavioral phenotype of AAV9-α1ACT-Q33 mice, we used arotarod performance test, an open-field assay, and a gait stabilityassessment using a video-assisted computerized treadmill (DigiGait) forgait analysis. AAV9-α1ACT-Q33 mice grew at roughly the same rate and hada comparable body weight to AAV9-α1ACT-Q11 and AAV9-GFP mice (FIG. 17A).However, AAV9-α1ACT-Q33 mice showed defects in motor functions asassessed by the rotarod test (FIG. 17B), activity in the open-fieldassay (FIG. 17C), and gait stability by the gait analysis assay (FIG.17D-FIG. 17E). AAV9-α1ACT-Q33 caused impaired performance on theaccelerating rotarod test in mice beginning at 4 weeks of age (P<0.01)(FIG. 17B). Ambulatory distance in the open-field test was significantlyshorter in AAV9-α1ACT-Q33 mice than in AAV9-α1ACT-Q11 and AAV9-GFP mice(P<0.01) (FIG. 17C). DigiGait analyses revealed that AAV9-α1ACT-Q33 miceexhibited shorter stride length (P<0.05) (FIG. 17D) and greater stridefrequencies (P<0.05) (FIG. 17E) in all four limbs from 4 weeks of age,indicating early-onset instability during walking. AAV9-α1ACT-Q33 miceoften showed weaving from side to side on a treadmill, whereasAAV9-α1ACT-Q11 and AAV9-GFP mice typically walked straight (movies 51 toS3). Furthermore, the deficit in behavioral phenotypes of AAV9-α1ACT-Q33mice and continuous CACNA1A IRES-driven α1ACT expression in thecerebellum were observed over a long-term follow-up of 30 weeks (FIG.26A to FIG. 26G). These findings indicate that AAV9-mediated somaticgene transfer of α1ACTSCA6 under the control of CACNA1A IRES causedsevere ataxia in mice at much earlier ages and more reproducibly thanthe Purkinje cell-specific promoter-driven transgenic SCA6 mouse modelwe previously developed (11).

miR-3191-5p Inhibits the CACNA1A IRES-Driven Translation of α1ACT whileSparing α1A and CACNA1A mRNA Expression

To develop a CACNA1A IRES-directed therapeutic approach for SCA6, weused an miRNA-mediated approach. miRNAs have been increasinglyrecognized to play a role in the regulation of gene expression, in manycases by both translational repression and mRNA destabilization (16,17). To date, miRNAs have not been used to preferentially regulate thetranslation of disease genes driven by a cellular IRES. Althoughprevailing evidence has pointed to the role of natural miRNAs in genesilencing and translational repression through binding to 3′untranslated regions (UTRs), or rarely 5′UTR, of targeted mRNAs (16-21),predicted miRNA binding sites are found throughout the genome includingin both coding and noncoding regions (22, 23).

We used the miRNA_Targets program (22) to predict that miRNAs-miR-711(accession number, MIMAT0012734), miR-3191-5p (accession number,MIMAT0022732), and miR-4786-3p (accession number, MIMAT0019955)—bind tosequences within the stem-loop structure of CACNA1A IRES (FIG. 27A toFIG. 27D) (24). To examine the effects of these miRNAs on CACNA1AIRES-driven translation, we cotransfected these miRNAs with either abicistronic reporter vector bearing CACNA1A IRES or a control vectorinto human embryonic kidney (HEK) 293 cells (FIG. 18A). Dual-luciferaseassays revealed that miR-711, miR-3191-5p, and miR-4786-3pdown-regulated CACNA1A IRES-driven luciferase activities compared to anegative control miRNA (FIG. 18B). We tested the effects of these miRNAson CACNA1A-encoded C-terminal FLAG-tagged peptides (α1A-FLAG andα1ACT-FLAG) with the normal (Q11) or pathological (Q33) polyQ tract intransfected HEK293 cells. Although miR-711 and miR-4786-3p decreasedfull-length α1A-FLAG and α1ACT-FLAG expression, miR-3191-5pdown-regulated α1ACT-FLAG expression but spared α1A-FLAG expression(FIG. 18C and FIG. 18D). This was the case with α1A-FLAG and α1ACT-FLAGin mice with the normal (Q11) or pathological (Q33) polyQ tract. qRT-PCRstudies of total RNA showed that miR-711 and miR-4′786-3p decreasedCACNA1A mRNA expression relative to a negative control miRNA, butmiR-3191-5p did not affect either CACNA1A-Q11 or CACNA1A-Q33 mRNAexpression (FIG. 18E and FIG. 18F). We also found that miR-3191-5p didnot affect endogenous CACNA1A mRNA expression in HEK293 cells (FIG.18G).

To determine whether miR-3191-5p interacts with regions within theCACNA1A mRNA other than the CACNA1A IRES, we prepared mutated CACNA1AIRES templates, resistant to the binding of miR-3191-5p (CACNA1AIRESmut) (FIG. 28A to FIG. 28C). CACNA1A IRESmut functioned normallyboth in the CACNA1A IRESmut dual-luciferase assays and in the α1A-FLAG-and α1ACT-FLAG-expressing HEK293 cells, but inhibition of expression bymiR-3191-5p was prevented (FIG. 28D and FIG. 28E). These results suggestthat miR-3191-5p interacts with CACNA1A IRES only through its predictedbinding site and inhibits the CACNA1A IRES-driven translation of α1ACTwhile sparing α1A expression and CACNA1A mRNA expression.

Ago4 is Required for miR-3191-5p-Mediated Inhibition of α1ACTTranslation

miRNAs generally act on targeted mRNAs in collaboration with themiRNA-induced silencing complex (miRISC)-guided Argonaute (Ago) proteins(25-29). To examine the role of Ago proteins on the inhibitory effectsof miR-3191-5p, we cotransfected HEK293 cells with miR-3191-5p and smallinterfering RNAs (siRNAs) targeting Ago1 to Ago4 carried on either abicistronic reporter vector bearing the CACNA1A IRES or a controlvector. Dual-luciferase assays revealed that knockdown of Ago4, but notAgo1 to Ago3, reversed the silencing effects of miR-3191-5p on theCACNA1A IRES-driven luciferase activities (FIG. 19A). Western blotanalyses showed that silencing of Ago4, but not Ago1 to Ago3, alsoprevented the down-regulation of α1ACT-FLAG expression by miR-3191-5p,suggesting that Ago4 is required for miR-3191-5p-mediated inhibition ofα1ACT translation (FIG. 19B).

To determine whether Ago4 directly associates with both miR-3191-5p andCACNA1A mRNA or whether Ago4 indirectly mediates the silencing effectsof miR-3191-5p, we performed RNA immunoprecipitation-coupled qRT-PCRstudies using antibodies specific to Ago1 to Ago4. We found that Ago2-and Ago4-specific antibodies preferentially precipitated miR-3191-5p toa greater extent than did control IgG. Only Ago4-specific antibodiesspecifically precipitated CACNA1A mRNA, indicating that Ago4 selectivelybound to both miR-3191-5p and CACNA1A mRNA (FIG. 19C and FIG. 19D).Ago1- and Ago3-specific antibodies had no effect. We also confirmed thatknockdown of Ago4 did not affect miR-3191-5p expression in HEK293 cells(FIG. 19E). These results suggest that miR-3191-5p bound to Ago4directly interacts with the CACNA1A IRES and selectively inhibits theCACNA1A IRES-driven α1ACT translation.

miR-3191-5p Inhibits the Translational Initiation of CACNA1A IRES-Drivenα1ACT by eIF4AII and eIF4GII

Because both eukaryotic initiation factors (eIFs) eIF4A and eIF4G havebeen previously shown to be involved in IRES-dependent translation inseveral types of viral IRESs (17), we examined the effects of these onCACNA1A IRES-driven α1ACT translation. We found that overexpression ofeither eIF4AII or eIF4GII, but not eIF4AI or eIF4GI, increased theCACNA1A IRES-driven luciferase activities and that the effects ofeIF4AII and eIF4GII were blocked by cotransfection with miR-3191-5p(FIG. 20A to FIG. 20D). Western blot analyses showed that overexpressionof either eIF4AII or eIF4GII increased both α1A-FLAG and α1ACT-FLAGexpression and that miR-3191-5p reversed the up-regulating effects ofeIF4AII and eIF4GII on α1ACT-FLAG expression without affecting eIF4AIIand eIF4GII expression (FIG. 20E and FIG. 20F). We also found thatoverexpression of either eIF4AII or eIF4GII in the presence or absenceof miR-3191-5p did not affect CACNA1A mRNA expression (FIG. 20G and FIG.20H). When we used CACNA1A IRESmut templates, the miR-3191-5p-mediatedinhibition of the up-regulating effects of eIF4AII and eIF4GII on bothCACNA1A IRESmut dual-luciferase activities and α1ACT-FLAG expressionwere prevented, indicating that the effects of miR-3191-5p depended onits interactions with CACNA1A IRES (FIG. 29A to FIG. 29D).

Because both eIF4AII and eIF4GII affected not only CACNA1A IRES-driventranslation of α1ACT but also cap-dependent translation of full-lengthα1A (FIG. 20E and FIG. 20F), we used IRES-α1ACT vectors (FIG. 24A) totest the binding affinities of eIF4AII and eIF4GII to CACNA1A IRES. RNAimmunoprecipitation-coupled qRT-PCR studies revealed that both eIF4AII-and eIF4GII-specific antibodies precipitated IRES-α1ACT mRNAs to agreater extent than did control IgG, and that binding affinities betweenIRES-α1ACT mRNAs and both eIF4AII and eIF4GII were decreased bytreatment with miR-3191-5p (FIG. 20I and FIG. 20J).Coimmunoprecipitation studies revealed that eIF4AII associated witheIF4GII (FIG. 20K). We also found that knockdown of eIF4GII had agreater effect on the down-regulation of CACNA1A IRES-driven α1ACTtranslation than did knockdown of eIF4AII (FIG. 20L). On the basis ofthese findings, we concluded that the complex of eIF4AII and eIF4GIIdirectly acts on the CACNA1A IRES to enhance IRES-driven α1ACTtranslation (FIG. 29E). miR-3191-5p bound to Ago4 selectively inhibitsthe translational initiation of CACNA1A IRES-driven α1ACT by eIF4AII andeIF4GII (FIG. 29F).

miR-3191-5p Prevents Purkinje Cell Degeneration in Mice Caused byCACNA1A IRES-driven α1ACTSCA6

To examine the therapeutic effects of miR-3191-5p-mediated reduction ofCACNA1A IRES-driven α1ACTSCA6 translation on the disease phenotype ofSCA6 mice, we constructed an AAV9 vector that allowed for thesimultaneous expression of GFP and either miR-3191-5p (AAV9-miR-3191-5p)or a nonspecific mock miRNA (AAV9-miR-mock) (FIG. 30A). We thenco-injected AAV9-α1ACT-Q33 with either AAV9-miR-3191-5p(AAV9-Q33-miR-3191-5p) or AAV9-miR-mock (AAV9-Q33-miR-mock) into theright lateral ventricle of neonatal wild-type C57/BL6J mice at postnatalday 1. Four weeks after AAV9 vector injection, we sacrificedAAV9-injected mice and found widespread transduction of miR-mock andmiR-3191-5p throughout the brain and cerebellum (FIG. 21A and FIG. 21C).We also found a high efficiency (>80%) of cotransduction ofAAV9-α1ACT-Q33 and either AAV9-miR-mock or AAV9-miR-3191-5p in thecerebellum of AAV9-injected mice (FIG. 30B and FIG. 30C). There was adecrease in CACNA1A IRES-driven α1ACT-Q33-FLAG protein expression in thebrain and cerebellum of AAV9-Q33-miR-3191-5p mice as compared toAAV9-Q33-miR-mock mice (FIG. 21B and FIG. 21D). Whereas IRES-α1ACT-Q33mRNA expression in the cerebellum of AAV9-Q33-miR-3191-5p mice wascomparable to that for AAV9-Q33-miR-mock mice (FIG. 21E), Western blotanalyses supported the silencing effect of miR-3191-5p on CACNA1AIRES-driven α1ACT-Q33-FLAG protein expression in the cerebellum ofAAV9-Q33-miR-3191-5p mice compared to AAV9-Q33-miR-mock mice (FIG. 21F).Using RNA immunoprecipitation-coupled qRT-PCR studies, we also confirmedthat Ago4 bound to both miR-3191-5p and IRES-α1ACT-Q33 mRNA in thecerebellum of AAV9-Q33-miR-3191-5p mice (FIG. 21G and FIG. 21H).

Immunohistochemical examination of AAV9-Q33-miR-3191-5p mouse cerebellumshowed that the treatment with AAV9-miR-3191-5p protected Purkinje cellsfrom degenerative changes (FIG. 22A to FIG. 22D). As compared toAAV9-Q33-miR-mock mice, AAV9-miR-3191-5p mice showed protection fromthinning of the molecular layer of the cerebellum (P<0.01) (FIG. 22B),decreased density of the dendritic tree (P<0.01) (FIG. 22C), anddecreased number of Purkinje cells (P<0.01) (FIG. 22D) caused byAAV9-delivered IRES-driven α1ACT-Q33. These results indicate thatAAV9-delivered miR-3191-5p inhibited Purkinje cell degeneration causedby CACNA1A IRES-driven α1ACTSCA6 in mice.

miR-3191-5p Prevents the Ataxia and Motor Deficits Caused by CACNA1AIRES-Driven α1ACTSCA6 in Mice

We further examined the therapeutic effect of AAV9-miR-3191-5p on mousebehavioral phenotypes caused by CACNA1A IRES-driven α1ACTSCA6. Althoughthe treatment with AAV9-miR-3191-5p did not affect the body weight ofAAV9-Q33-miR-3191-5p mice compared to AAV9-Q33-miR-mock mice (FIG. 23A),we found that the CACNA1A IRES-driven α1ACTSCA6-associated diseasephenotypes in AAV9-Q33-miR-3191-5p mice were prevented, as assessed bythe rotarod test (FIG. 23B), open-field assay (FIG. 23C), and DigiGaitfor gait analysis (FIG. 23D and FIG. 23E). AAV9-Q33-miR-3191-5p miceperformed significantly better on the accelerating rotarod test (P<0.01)(FIG. 23B) and ambulated a greater distance in the open-field test thandid AAV9-Q33-miR-mock mice (P<0.01) (FIG. 23C) at 4 weeks of age. Ascompared to AAV9-Q33-miR-mock mice, AAV9-Q33-miR-3191-5p mice exhibitedimprovement of gait instability in all four limbs caused byAAV9-delivered IRES-driven α1ACT-Q33 (P<0.05) (FIG. 23D and FIG. 23E).We also found that treatment with AAV9-miR-3191-5p prevented the weavingsteps of AAV9-Q33-miR-3191-5p mice (movies S4 and S5). Furthermore, thetherapeutic effect of AAV9-miR-3191-5p on mouse behavioral phenotypesand CACNA1A IRES-driven α1ACT-Q33 expression in the cerebellum ofAAV9-Q33-miR-3191-5p mice persisted during long-term follow-up of 30weeks (FIG. 31A to FIG. 31G).

Although both mature and stem-loop sequences of hsa-miR-3191-5p used inour study are identified in the human genome (miRBase:www.mirbase.org/), those of mmu-miR-3191-5p have not yet been identifiedin the mouse genome. To identify other potential candidates ofhsa-miR-3191-5p-targeting mRNAs in mouse, we first used TargetScanHuman7.0 (www.targetscan.org/vert70/) to find the top 100 rankedhsa-miR-3191-5p-targeting human mRNAs. We selected 13 genes with highreliability (cumulative weighted context++ score<−1 and total context++score<−1) (FIG. 32A). Among these, seven genes (PRX, ZNF781, C22orf46,ZNF23, ZNF286A, ERBB4, and PTBP1) have hsa-miR-3191-5p binding siteswithin the 3′UTR of their conserved mouse orthologs. We examined themRNA levels of these seven genes in the cerebellum of wild-type miceinjected with AAV9-miR-3191-5p compared to those injected withAAV9-miR-mock. We found that AAV9-miR-3191-5p down-regulated the mRNAexpression of mouse orthologs of ZNF781, C₂₂orf46, ZNF23, and ERBB4 byabout 50%, whereas those of the other three genes were unchanged,indicating that not all of the predicted mRNA targets were affected bythe treatment with AAV9-miR-3191-5p in mice (FIG. 32B to FIG. 32H).

To determine whether an overall change in whole-gene expression in themouse brain and cerebellum after AAV9-miR-3191-5p injections leads tosignificant adverse and toxic effects, we monitored wild-type miceinjected with AAV9-miR-3191-5p for 12 months and found no abnormalphenotypes. Also, we found no histopathological abnormalities in thebrain, cerebellum, heart, lung, liver, and kidney of wild-type miceinjected with AAV9-miR-3191-5p (FIG. 33A to FIG. 33F). On the basis ofthese findings, we conclude that AAV9-mediated delivery of miR-3191-5pis a well-tolerated and successful therapeutic approach for treating thedisease phenotype in our SCA6 mouse model.

Discussion

To explore possible therapeutic strategies for treating SCA6, wedeveloped a robust SCA6 mouse model using AAV9-mediated somatic genetransfer of the CACNA1A second cistron to express α1ACTSCA6 under thecontrol of CACNA1A IRES. We found prominent α1ACTSCA6 expression thatwas predominantly in cerebral cortex and cerebellum of AAV9-injectedmice. Mice injected with AAV9 expressing α1ACTSCA6 showed unambiguousdisease phenotypes associated with the pathological and clinicalfeatures of patients with SCA6 (2-8) much earlier and more reproduciblythan the Purkinje cell-specific promoter-driven transgenic SCA6 mousemodel we previously developed (11) or the knock-in SCA6 mouse model withpolyQ expansion size observed in patients with SCA6 (9, 10). Byimmunofluorescence analysis, we found that administration of a total of1010 vg of AAV9 expressing CACNA1A IRES-driven α1ACTSCA6 into thelateral ventricle caused an -50% decrease in the number of Purkinjecells that was sufficient to lead to early-onset ataxia and motordeficits from an early age. These findings indicate that AAV9-mediatedCACNA1A IRES-driven α1ACTSCA6 expression is stronger and therefore moretoxic in mouse cerebellum than α1ACTSCA6 expression in previouslydeveloped SCA6 mouse models (9-11). These early and severe ataxicphenotypes are also observed in Pumiliol-deficient mice, mimicking SCA1,another type of autosomal dominant SCA (30).

Although we have previously shown that α1ACT functions as atranscription factor essential for cerebellar development (11), thedetailed mechanism by which α1ACTSCA6 causes Purkinje cell degenerationin adulthood remains unknown. Because most individuals with SCA6 areheterozygous for the SCA6-causing expansion, that is, they still haveone copy of the normal CACNA1A second cistron, they express some levelsof the normal α1ACT protein. On the other hand, SCA6 homozygotes thatexpress two copies of mutant α1ACT protein and no normal α1ACT proteindevelop normally and have nearly the same age of onset as SCA6heterozygotes (7, 8, 31-33). This confirms that the mutant α1ACT proteinfulfills some of the normal α1ACT protein functions in development andthat SCA6 arises by a dominant “gain-of-function” mechanism.

In cell culture experiments, we have discovered that miR-3191-5pdirectly acts on the CACNA1A IRES to regulate the translation of α1ACTin an Ago4-dependent manner. Previous studies have shown that miRNAs, byforming miRISC with Ago1 or Ago2, block the assembly of translationinitiation factors to repress either cap- or IRES-dependent translationthrough their binding to the 3′UTR of targeted mRNA (18-20). There arefour members of the RNA-induced silencing complex-related Ago proteinfamily in human Ago1, Ago2, Ago3, and Ago4 (27). Very little is knownabout Ago4, although the absence of a catalytic domain suggests that itmight have a distinct role in gene silencing compared with Ago2 (34-39).Our data showing that Ago4 interacts with miR-3191-5p and CACNA1A IRESto repress translation of the second cistron may suggest that it has aspecific role for the mRNA-sparing translational repression via miRISC,possibly even selectively for the translational repression of IRESs.

We have also demonstrated that miR-3191-5p bound to Ago4 selectivelyinhibits the translational initiation of α1ACT by eIFs, eIF4AII andeIF4GII, which enhance α1ACT expression. Although the list of cellularmRNAs that are thought to contain IRESs is growing (17, 40-42), cellularIRESs show little structural relationship to each other, and theirunderlying mechanism remains largely unknown but is thought to followthe picornavirus paradigm of binding to the eIF4A-eIF4G complex (17).Identification of eIF4AII, which may provide helicase activity, andeIF4GII, which may serve as a scaffold (17, 43-45), in the mechanism ofCACNA1A IRES-mediated translation supports recent findings that show theeffect of eIFs on cellular IRESs in human disease (40-42). Presumably,in addition to these eIFs, other IRES transacting factors and small RNAsmay play a part in the regulation of CACNA1A IRES-mediated translation(17, 40, 42). The recently recognized abundance of IRES-mediatedtranslational mechanisms in the human genome raises the potential fortheir role in human disease (46).

We have developed an miRNA-mediated therapy for SCA6 using selectivetranslational blockade of the CACNA1A second cistron in mice. Ourfindings indicate that continuous inhibition of α1ACTSCA6 expression bythe AAV9 vector-mediated delivery of miR-3191-5p has a substantialtherapeutic effect on the SCA6 disease phenotype in mice. Consideringthat an -50% decrease in the number of Purkinje cells is sufficient tocause ataxia and motor deficits in our mouse model, miRNA-mediatedα1ACTSCA6 silencing by blockade of CACNA1A IRES-driven translation ingreater than half of Purkinje cells would have great potential benefitin presymptomatic or symptomatic SCA6 patients (13-15).

Although AAV9-mediated intervention with miR-3191-5p did not regulateall of the predicted mRNA targets in our mouse model, we showed thatmiR-3191-5p has a selective silencing effect on the CACNA1A IRES-driventranslation of α1ACTSCA6 in mice. Targeting CACNA1A IRES in SCA6patients through miRNAs or small molecules may down-regulate both normaland mutant α1ACT. However, AAV9-mediated administration of miR-3191-5pinto the lateral ventricle did not lead to any toxic effects in miceduring long-term observations over 1 year. Other studies in our groupalso showed that the effect of α1ACT on establishing normal cerebellardevelopment appeared to be irreversible (47).

There are several limitations to our study. Although patients with SCA6develop ataxic phenotypes in adulthood (2-8), the targeted expression ofα1ACTSCA6 in late adult mouse cerebellum has not been evaluated. Also,the off-target effects due to viral transduction of an miRNA may dependon viral titer and differ among species. A series of reports havesuggested that miRNAs may have therapeutic promise for treating cancer,metabolic disease, and inflammation (48-50). However, before miRNAs canhave success as therapeutic agents, many challenges remain to beovercome, including their efficient delivery and off-target effects (51,52). Further studies are needed to validate more efficient and specifictargeting strategies to develop an miRNA-based therapy for SCA6 patientsin the future.

Although therapy targeting IRES activity has been a mainstay ofantiviral therapy, it has not been used for genetic diseases (53-55).IRES-dependent translation also plays a role in the expression of someoncogenes, such as c-myc (56), L-myc (57), and epidermal growth factorreceptor (58), in tumors. Our study opens the door to the development oftherapies using a new strategy for the selective suppression ofIRES-driven pathogenic gene products based on delivery ofdisease-specific miRNAs.

Example 4

The following example describes the materials and methods used in thestudy of Example 10.

Study Design

Our study was based on our previous findings that SCA6 is attributableto a polyQ repeat expansion within a second CACNA1A gene product, α1ACT,and that α1ACT expression is under the control of an IRES present withinthe CACNA1A coding region. The objectives of the current study werethreefold. The first objective was to develop an early-onset ataxiamodel using an AAV9-based gene delivery system to express CACNA1AIRES-driven α1ACTSCA6. The second objective was to identify miRNAs thattarget CACNA1A IRES and preferentially block the CACNA1A IRES-driventranslation of α1ACT. Finally, we investigated the therapeutic potentialof ectopic miRNA expression for the treatment of SCA6 by AAV9-mediatedtransduction in our previously developed mouse model of CACNA1AIRES-driven SCA6. On the basis of our previous studies (11, 59), four tosix biological replicates were used for each in vitro or in vivobiochemical and histological analysis, whereas a sample size of 12 mice(6 male and 6 female mice) per group was used for behavioral testing.For AAV9 injections, neonatal mice were randomly assigned to treatmentconditions with equivalent numbers in each group. All behavioralanalyses were performed by experimenters who were blind to the identityof treatment conditions. Data collection and the biochemical andhistological analysis for mouse samples were performed with theinvestigators unaware of the sample identities until statisticalanalyses. All source data are in the Supplementary Materials (table 51).

Construction of DNA Plasmids

We used pcDNA3 vectors expressing CACNA1A-encoded FLAG-tagged peptides(α1A-FLAG and α1ACT-FLAG), a bicistronic CACNA1A IRES reporter vector,and a control reporter vector as described previously (11). Weconstructed the truncated transgenes of CACNA1A second cistron thatlacked the sequence of 5′ upstream from CACNA1A IRES (pcDNA3-IRES-α1ACTvectors; FIG. 24A) and inserted them into AAV9 genomes to prepareAAV9-α1ACT-Q11 and AAV9-α1ACT-Q33 (FIG. 24B). Briefly, the sequence ofnucleotides 4962 to 7757 of full-length CACNA1A complementary DNA (cDNA)(NM_001127222.1), corresponding to the sequence of CACNA1A IRES andα1ACT ORF, was amplified using IRES-α1ACT primers[5′-ATCAGGATCCGCCCTCAACACCATCGTGC-3′(forward)(SEQ ID NO: 186) and5′-GAATCTAGATTACTTGTCATCG-3′ (reverse) (SEQ ID NO: 187)] from pcDNA3vectors expressing CACNA1A-encoded FLAG-tagged peptides. The PCRproducts were inserted into the Bam HI and Xba I sites of pcDNA3vectors. We also modified the sequence of nucleotides 5013 to 5015 from“TAT” to “TAG” of stop codon (FIG. 24A).

To obtain mutated CACNA1A IRES transcripts that are resistant to thebinding of miR-3191-5p (CACNA1A IRESmut vectors; FIG. 28B and FIG. 28C),we performed C-G and G-C substitutions within the predicted miR-3191-5pbinding site. We also performed C-G and G-C substitutions within thecomplementary binding site of the sequence targeted by miR-3191-5p tomaintain the stem-loop structure of CACNA1A IRESmut as same as that ofthe original. Briefly, we amplified the predicted binding site ofmiR-3191-5p using miR mut primers[5′-ATGTCTCCGCCCCTGGGTCTccccAAcAAcTcTggccCCAGAGTGGCTTACAAGCG-3′(forward) (SEQ ID NO: 182) and5′-CGCTTGTAAGCCACTCTGGggccAgAgTTgTTggggAGACCCAGGGGCGGAGACAT-3′(reverse)(SEQ ID NO: 183)] and the complementary binding site of thesequence targeted by miR-3191-5p using anti-miR mut primers[5′-ACTGAGCACAATAACTTggccAggTTgTTggAGGCCCTCATGCTTCTC-3′ (forward)(SEQ IDNO: 184) and 5′-GAGAAGCATGAGGGCCTccAAcAAccTggccAAGTTATTGTGCTCAGT-3′(reverse)(SEQ ID NO: 185)]. Subsequently, we annealed PCR products usingprimers anti-miR mut forward and miR mut reverse and inserted theannealed PCR products into the BIp I sites of pcDNA3 vectors expressingCACNA1A-encoded FLAG-tagged peptides and into the Eco RI and Nco I sitesof a bicistronic CACNA1A IRES reporter vector.

pDEST-GFP-Ago1 (Addgene, plasmid #21534) and pDEST-GFP-Ago4 (Addgene,plasmid #21536) were gifts from E. Chan (60). pEGFP-hAgo2 (Addgene,plasmid #21981) was a gift from P. Sharp (61). pGEX-GST-AGO3 (Addgene,plasmid #24318) was a gift from C. Novina (34). To create pEGFP-AGO3, werestriction-digested pGEX-GST-AGO3 with Eco RI and Bam HI and insertedit into the plasmid backbone of the pEGFP-hAgo2 after Eco RI and Bam HIdigestion.

pcDNA3-HA-eIF4GI and pcDNA3-HA-eIF4GII were gifts from N. Sonenberg(McGill University, Canada) (62). M. Bushell (Medical Research Council,UK) provided us with the plasmids expressing human eIF4AI and eIF4AII(18). pcDNA3.1-HisXpress vector was a gift from T. Cooper (BaylorCollege of Medicine). From the plasmids expressing human eIF4AI andeIF4AII, we amplified human eIF4AI and eIF4AII cDNAs using eIF4AI-PCRprimers [5′-ATCAGGATCCATGTCTGCGAGCCAGGAT-3′ (forward)(SEQ ID NO: 188)and 5′-ATCAGGGCCCAGGTCAGCAACATTGAGG-3′ (reverse)(SEQ ID NO: 189)] andeIF4AII-PCR primers [5′-ATCAGGATCCATGTCTGGTGGCTCCGCG-3′ (forward)(SEQ IDNO: 190) and 5′-ATCAGGGCCCTTAAATAAGGTCAGCCAC-3′ (reverse)(SEQ ID NO:191)], respectively. Subsequently, we inserted the PCR products into BamHI- and Apa I-digested pcDNA3.1-HisXpress vectors to obtainpcDNA3.1-HisXpress-eIF4AI and pcDNA3.1-HisXpress-eIF4AII. We used theoriginal, either pcDNA3.1-HisC or pcDNA3-HA, vectors lacking theinsertions in the multiple cloning sites as control vectors.

Cotransfection of DNA Plasmids with Either Synthetic miRNA or siRNA intoCultured Cells

HEK293 cells were cultured in Dulbecco's modified Eagle's mediumsupplemented with 10% fetal bovine serum. All miRNAs and siRNAs werepurchased from Life Technologies. ID numbers are shown as follows:miR-711 (MC15715), miR-3191-5p (MC23769), miR-4786-3p (MC21295), anegative control miRNA (#4464059), Ago1 (s25500), Ago2 (s25931), Ago3(s46947), Ago4 (s46949), eIF4AII (s4570), eIF4GII (s16519), and an siRNAnegative control (#12935-300).

We plated HEK293 cells onto six-well dishes and cotransfected each dishwith 1.0 μg of the vector expressing the following: bicistronic CACNA1AIRES reporter, bicistronic CACNA1A IRESmut reporter, control reporter,and α1A-FLAG and α1ACT-FLAG; 0.5 μg of the vector expressing thefollowing: eIF4AI, eIF4AII, eIF4GI, and eIF4GII; and either 20 nMsynthetic miRNA or 20 nM siRNA molecules. We used Lipofectamine 2000(Life Technologies) as a transfection reagent in all cases. Neither themiRNA negative control nor the siRNA negative control matched any humanmRNA. The transfected cells were cultured for 48 hours before beingprocessed for RNA and protein analysis.

Luciferase Assay

HEK293 cells were plated onto six-well dishes and cotransfected withvectors as shown above. Forty-eight hours after cotransfection, theactivities of firefly and Renilla luciferase in lysates prepared fromtransfected cells using the Dual-Luciferase Assay System (Promega) weremeasured by using a Wallac 1420 VICTORS V luminometer with a 1-sintegration time (PerkinElmer). The ratio of firefly luciferase toRenilla luciferase activities of a bicistronic CACNA1A IRES reportervector was normalized to that of a bicistronic control reporter vector.

Protein Expression Analysis

We did Western blot analysis as previously described (11, 63). We usedthe following primary antibodies: FLAG-specific antibody (1:5000, A8592and 1:5000, F1804; Sigma-Aldrich); Ago1-specific antibody (1:2000,9388S; Cell Signaling Technology); Ago2-specific antibody (1:2000,SAB4200085; Sigma-Aldrich); Ago3-specific antibody (1:2000, 5054S; CellSignaling Technology); Ago4-specific antibody (1:2000, 6913S; CellSignaling Technology); eIF4AII-specific antibody (1:2000, ab31218;Abcam); eIF4GII-specific antibody (1:1000, sc-100732; Santa CruzBiotechnology); GFP-specific antibody (1:2000, M048-3; MBL); andGAPDH-specific antibody (1:5000, AM4300; Life Technologies). We used NIHImageJ software to quantify the specific expression of individualproteins and demonstrated the relative signal intensities of individualproteins normalized to those of GAPDH in each sample.

Quantitative Real-Time Polymerase Chain Reaction

The total RNA was extracted from HEK293 cells and the cerebellum of miceusing the miRNeasy Mini Kit (Qiagen) and reverse-transcribed using theSuperScript VILO (Life Technologies) for mRNA and NCode VILO (LifeTechnologies) for miRNA. The cDNAs were then used for real-time PCRusing the iQ SYBR Green Supermix (Bio-Rad Laboratories). We did theamplification, detection, and data analysis using a Bio-Rad iCyclersystem (Bio-Rad Laboratories). The crossing threshold values for themRNAs of the individual genes were normalized to β-actin. The crossingthreshold values for miR-3191-5p were normalized to U6 small nuclearRNA. Changes in the expression of mRNA and miRNA were expressed as afold change relative to the control. We used the following primers. Thesequences of the hsa-CACNA1A primers were 5′-GTCTGGGGAAGAAGTGTCCG-3′(forward) (SEQ ID NO: 192) and 5′-GCTCCTCCCTTGGCAATCTT-3′ (reverse) (SEQID NO: 193). These hsa-CACNA1A primers discriminated between the humanCACNA1A mRNA and the mouse CACNA1A mRNA. The sequences of thehsa-IRES-α1ACT primers were 5′-GTACCTCACCCGAGACTCCT-3′ (forward) (SEQ IDNO: 207) and 5′-CGGACACTTCTTCCCCAGAC-3′ (reverse) (SEQ ID NO: 208).These primers can also detect the mouse endogenous CACNA1A mRNA. Thesequences of the hsa-β-actin primers were 5′-GCGGGAAATCGTGCGTGACATT-3′(forward) (SEQ ID NO: 196) and 5′-GATGGAGTTGAAGGTAGTTTCGTG-3′ (reverse)(SEQ ID NO: 197). The sequences of the mmu-Prx primers were5′-TTGGTGGAGATTATCGTGGAG-3′ (forward) (SEQ ID NO: 209) and5′-TCTTGCAAGCTGAGGCTCTTA-3′ (reverse) (SEQ ID NO: 210). The sequences ofthe mmu-Zfp781 primers were 5′-CCTATGAGGATGTGCATGTGA-3′ (forward) (SEQID NO: 211)and 5′-TGGGGTCCAGAGTGACAGATA-3′ (reverse) (SEQ ID NO: 212).The sequences of the mmu-4930407I10Rik primers were5′-GAATTCCCAAGGGCTAAAGTG-3′ (forward) (SEQ ID NO: 213)and5′-TTTCACACCATCTCCACTTCC-3′ (reverse) (SEQ ID NO: 214). The sequences ofthe mmu-Zfp612 primers were 5′-AAGGCAGCCCTCAAGTTAATC-3′ (forward) (SEQID NO: 215)and 5′-AAGTCTCTGATGCCAGACGAA-3′ (reverse) (SEQ ID NO: 216).The sequences of the mmu-Zfp286 primers were 5′-CATGGAAACCAGACCTGAGAG-3′(forward) (SEQ ID NO: 217) and 5′-ACGCTCACATTCAAGAGCAGT-3′ (reverse)(SEQ ID NO: 218). The sequences of the mmu-Erbb4 primers were5′-CGCTAGAACTCCACTGATTGC-3′ (forward) (SEQ ID NO: 219). and5′-TACCAGCTCTGTCTCCAGGAA-3′ (reverse) (SEQ ID NO: 220). The sequences ofthe mmu-Ptbp1 primers were 5′-TCACCAAGAACAACCAGTTCC-3′ (forward) (SEQ IDNO: 221)and 5′-GTGAGCTTGGAGAAGTCGATG-3′ (reverse) (SEQ ID NO: 222). Thesequences of the mmu-f3-actin primers were 5′-GCTACAGCTTCACCACCACA-3′(forward) (SEQ ID NO: 198) and 5′-TCTCCAGGGAGGAAGAGGAT-3′ (reverse) (SEQID NO: 199). The sequences of the miR-3191-5p primers were5′-GCTCTCTGGCCGTCTAC-3′ (forward) (SEQ ID NO: 200)and5′-GTCCAGTTTTTTTTTTTTTTTGGAAG-3′ (reverse) (SEQ ID NO: 201). Thesequences of the U6 small nuclear RNA primers were5′-CTTCGGCAGCACATATACTAAA-3′ (forward) (SEQ ID NO: 202) and5′-AAAATATGGAACGCTTCACG-3′ (reverse) (SEQ ID NO: 203). We designed themiR-3191-5p primers using a bioinformatics program (64).

Coimmunoprecipitation

We plated HEK293 cells onto 100-mm dishes and cotransfected each dishwith 3.0 μg of the vectors expressing eIF4AII and eIF4GII. Forty-eighthours after transfection, we harvested HEK293 cells forimmunoprecipitation using eIF4AII-specific antibody (5 μg per sample,ab31218; Abcam), eIF4GII-specific antibody (5 μg per sample, sc-100732;Santa Cruz Biotechnology), and Dynabeads Protein G ImmunoprecipitationKit (Life Technologies) according to the manufacturer's suggestedprotocols. A rabbit IgG (5 μg per sample, PP64B; Millipore) and mouseIgG (5 μg per sample, CS200621; Millipore) were used as controls.

Immunoprecipitation-Coupled qRT-PCR

We plated HEK293 cells onto 100-mm dishes and cotransfected each dishwith 2.0 μg of the vector expressing the following: full-lengthCACNA1A-Q33-encoded FLAG-tagged peptides, IRES-α1ACT-Q33, miR-3191-5p(SC401396; OriGene), Ago1, Ago2, Ago3, Ago4, eIF4AII, and eIF4GII.Forty-eight hours after transfection, we harvested HEK293 cells forcoimmunoprecipitation using Ago1-specific antibody (5 μg per sample,9388S; Cell Signaling Technology), Ago2-specific antibody (5 μg persample, 2897S; Cell Signaling Technology), Ago3-specific antibody (5 μgper sample, 5054S; Cell Signaling Technology), Ago4-specific antibody (5μg per sample, 6913S; Cell Signaling Technology), eIF4AII-specificantibody (5 μg per sample, ab31218; Abcam), eIF4GII-specific antibody (5μg per sample, sc-100732; Santa Cruz Biotechnology), and Magna RIPRNA-Binding Protein Immunoprecipitation Kit (Millipore) according to themanufacturer's suggested protocols. A rabbit IgG (5 μg per sample,PP64B; Millipore) and mouse IgG (5 μg per sample, CS200621; Millipore)supplied by the manufacturer were used as controls. Theimmunoprecipitated RNA was reverse-transcribed using SuperScript VILO(Life Technologies) for mRNA and NCode VILO (Life Technologies) formiRNA and analyzed by qRT-PCR for the differential expression ofCACNA1A-Q33 mRNA and IRES-α1ACT-Q33 mRNA using the following primers:5′-GTCTGGGGAAGAAGTGTCCG-3′ (forward) (SEQ ID NO: 192) and5′-GCTCCTCCCTTGGCAATCTT-3′ (reverse) (SEQ ID NO: 193), and miR-3191-5p,5′-GCTCTCTGGCCGTCTAC-3′ (forward) (SEQ ID NO: 200) and5′-GTCCAGTTTTTTTTTTTTTTTGGAAG-3′ (reverse) (SEQ ID NO: 201). We alsoextracted RNA and protein complex from the cerebellum of AAV9-injectedmice and harvested them for coimmunoprecipitation as same as shownabove.

Development of the AAV9 Vectors

The AAV9 vector plasmids contained an expression cassette, consisting ofa human cytomegalovirus immediate-early promoter followed by cDNAencoding gene of our interest as shown below, woodchuck hepatitis virusposttranscriptional regulatory element (WPRE), and a simian virus 40polyadenylation signal sequence between the inverted terminal repeats ofthe AAV3 genome. The AAV9 vectors expressing α1ACT with either normalCAG repeat size (AAV9-α1ACT-Q11) or mutant CAG repeat size(AAV9-α1ACT-Q33) contained the truncated transgenes of CACNA1Acorresponding to the sequence of CACNA1A IRES and α1ACT ORF (IRES-α1ACT;FIG. 24A and FIG. 24B). AAV9-GFP contained cDNA encoding GFP sequence.AAV9-miR-3191-5p contained cDNA encoding GFP and miR-3191-5p sequence(FIG. 30A). AAV9-miR-mock contained cDNA encoding GFP and the miR-mocksequence (FIG. 30A). The sequences of miR-3191-5p and miR-mock are5′-GGGGTCACCTCTCTGGCCGTCTACCTTCCACACTGACAAGGGCCGTGGGGACGTAGCTGGCCAGACAGGTGACCCC-3′ (miR-3191-5p) (SEQ ID NO: 181) and5′-GTATTGCGTCTGTACACTCACCGTTTTGGCCACTGACTGACGGTGAGTGCAGACGCA ATA-3′(miR-mock) (SEQ ID NO: 206).

We synthesized the AAV9 vp cDNA as previously described with thesubstitution of thymidine for adenine 1337, which introduced an aminoacid change from tyrosine to phenylalanine at position 446 (65).Recombinant AAV9 vectors were produced by transient transfection intoHEK293 cells using the vector plasmid, an AAV3 rep and AAV9 vpexpression plasmid, and the adenoviral helper plasmid pHelper (AgilentTechnologies). We purified the recombinant viruses by isolation from twosequential continuous CsCl gradients, and the viral titers weredetermined by qRT-PCR as follows: 40 cycles of 95° C./15 s, 60° C./30 s,72° C./90 s, and 75° C./15 s with WPRE forward primer(5′-ATTGCTTCCCGTATGGCTTTCA-3′) (SEQ ID NO: 223) and WPRE reverse primer(5′-TCAGCAAACACAGTGCACACCA-3′) (SEQ ID NO: 224) to amplify the sequenceof nucleotides 1319 to 1201 of woodchuck hepatitis virus 2.

Injection of AAV9 into the Ventricle of Neonatal Wild-Type Mice

The C57/BL6J mice were purchased from Jackson Laboratory and maintainedin our breeding colony. At postnatal day 1, neonatal C57/BL6J mice wereindividually anesthetized on ice, and a total of 1010 vg in 2 to 4 μl ofAAV9 solution were injected into the right lateral ventricle of neonatalC57/BL6J mice with a 10-μl Hamilton syringe attached to a 32-gaugeneedle (Hamilton Company). The viral solution contained 0.04% trypanblue (Sigma-Aldrich) to help determine whether the ventricles wereindeed injected. Only those neonatal C57/BL6J mice in which the lateralventricles were filled with viral solution were analyzed. Six male andsix female mice were enrolled into each group: two groups of AAV9-GFPmice, AAV9-α1ACT-Q11 mice, AAV9-α1ACT-Q33 mice, AAV9-Q33-miR-mock mice,and AAV9-Q33-miR-3191-5p mice. All animal experiments were approved andcarried out in accordance with the regulations and guidelines for thecare and use of experimental animals at the Institutional Animal Careand Use Committee of the University of Chicago.

Injection of AAV9 into the Ventricle of Neonatal Wild-Type Mice

The C57/BL6J mice were purchased from Jackson Laboratory and maintainedin our breeding colony. At postnatal day 1, neonatal C57/BL6J mice wereindividually anesthetized on ice, and a total of 1010 vg in 2 to 4 μl ofAAV9 solution were injected into the right lateral ventricle of neonatalC57/BL6J mice with a 10-μl Hamilton syringe attached to a 32-gaugeneedle (Hamilton Company). The viral solution contained 0.04% trypanblue (Sigma-Aldrich) to help determine whether the ventricles wereindeed injected. Only those neonatal C57/BL6J mice in which the lateralventricles were filled with viral solution were analyzed. Six male andsix female mice were enrolled into each group: two groups of AAV9-GFPmice, AAV9-α1ACT-Q11 mice, AAV9-α1ACT-Q33 mice, AAV9-Q33-miR-mock mice,and AAV9-Q33-miR-3191-5p mice. All animal experiments were approved andcarried out in accordance with the regulations and guidelines for thecare and use of experimental animals at the Institutional Animal Careand Use Committee of the University of Chicago.

The Behavioral Assessments of AAV9-Injected Mice

We examined the behavioral assessments of AAV9-injected mice at 4, 8,12, and 30 weeks of age. The investigators who carried out thebehavioral assessments were blinded to the treatment conditions.

Rotarod. We analyzed rotarod test of mice using Economex Rotarod(Columbus Instruments) with accelerating mode (4 to 40 rpm, accelerationwith 0.1 rpm per 0.8 s). We performed three consecutive trials with10-min intervals between each trial.

Open-field assay. We examined open-field assay using Mouse Open FieldArena and 48 Channel IR Controller for Open Field Activity (ENV-510 andENV-520, Med Associates Inc.). Briefly, mice were placed in the centerof the open-field area, and their movements were monitored through theside-mounted photobeams for 30 min. We analyzed multiple parametersusing Activity Monitor software (Med Associates Inc.) and adopted totaldistance traveled to assess the activity of each mouse.

DigiGait analysis. We also examined a video-assisted computerizedtreadmill for mouse gait analysis using a DigiGait with DigiGaitsoftware (Mouse Specifics). All mice were tested at the speed of 25cm/s.

Immunohistochemistry, Immunofluorescence, and Histopathology

Immunofluorescence was performed as previously described (11, 59) exceptas modified below. We cut paraffin-embedded sagittally oriented 5-μmsections of mouse brains and cerebellums at 20-μm intervals. We usedcomparable sections from vermis, medial hemisphere, and lateralhemisphere for staining and histopathological assessments.Paraffin-embedded sections of perfused brains and cerebellums weredewaxed, rehydrated, and then steamed for 20 min in antigen retrievalsolution (Reveal, Biocare Medical). Sections were blocked and exposed toprimary antibodies for 12 hours at 4° C. After washing, fluorescentsecondary antibodies in phosphate-buffered saline and 0.05% Tween 20were added for 1 hour at room temperature. Confocal fluorescence imageswere captured with a Leica TCS SP2 laser scanning confocal microscope(Leica Microsystems Inc.).

We used NIH ImageJ software to quantify the FLAG and GFP expression ineach section and demonstrated the relative signal intensities of theFLAG and GFP fluorescence per unit area of the mouse hippocampus,cerebral cortex, and cerebellum in each section. The molecular layerthickness and density of Purkinje dendritic trees were calculated aspreviously described (11, 66). We selected Purkinje cells well stainedwith GFP- and FLAG-specific antibodies, indicating well transduced withAAV9, and analyzed Purkinje cells (100 to 250 cells per sample) in theentire area of each section to calculate the mean of the density ofPurkinje dendritic trees. The dendritic trees of the captured Purkinjecell image and the area enclosed were outlined and measured using NIHImageJ software. We also calculated the number of Purkinje cells (100 to250 cells per sample) in the entire area of each section and expressedthe results as the number per 250 μm.

We used the following primary antibodies: FLAG-specific antibody (1:200,F1804 and 1:200, F7425; Sigma-Aldrich), GFP-specific antibody (1:200,M048-3; MBL), and calbindin-specific antibody (1:200, CB38a; Swant).Goat Alexa Fluor-conjugated anti-mouse and goat Alexa Fluor-conjugatedanti-rabbit IgG antibodies (Life Technologies) were used for secondaryfluorescence detection. For the tissue sections stained with hematoxylinand eosin, digital image files were created with a 3D Histech PannoramicScan whole slide scanner (PerkinElmer) with a Stingray F146C colorcamera (Allied Vision Technologies). Individual images were analyzedusing the 3D Histech Pannoramic Viewer software (PerkinElmer).

Statistical Analysis

Statistical analysis was performed using ANOVA and Student's t test,unless specified, with the IBM SPSS Statistics 23.0. Two-tailed unpairedt test was used to compare two conditions. One-way ANOVA was used forcomparison among multiple experimental conditions. Bonferroni post hoctest was used when comparing among each condition. For the analysis ofmouse body weight and rotarod performance, two-way ANOVA was used forcomparison among groups. All data represent means±SEM. Statisticalsignificance in figures: *P<0.05, **P<0.01, ***P<0.001.

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All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range and each endpoint, unless otherwise indicatedherein, and each separate value and endpoint is incorporated into thespecification as if it were individually recited herein.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein, is intended merely to better illuminate theinvention and does not pose a limitation on the scope of the inventionunless otherwise claimed. No language in the specification should beconstrued as indicating any non-claimed element as essential to thepractice of the invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A method of treating a subject with spinocerebellar ataxia Type 6(SCA6) or with a predisposition to spinocerebellar ataxia Type 6 (SCA6),comprising the step of administering to the subject (i) an antisensemolecule, (ii) a vector encoding the antisense molecule, (iii) a cellcomprising the vector or antisense molecule, (iv) a extracellularvesicle comprising the antisense molecule, or (v) a combination thereof,wherein the antisense molecule binds to a portion of an IRES of aCACNA1A gene comprising the sequence of SEQ ID NO:
 180. 2. (canceled) 3.The method of claim 1, wherein the antisense molecule comprises thesequence of SEQ ID NO:
 179. 4. The method of claim 1, wherein the vectorcomprises a nucleotide or nucleotide analog sequence encoding a miRNAantisense molecule comprising the base sequence of SEQ ID NO: 179 or SEQID NO:
 181. 5-13. (canceled)
 14. The method of claim 1, wherein the cellor extracellular vesicle is autologous to the subject.
 15. A syntheticantisense molecule which specifically binds to a portion of an IRES of aCACNA1A gene comprising the sequence of SEQ ID NO: 180 comprising atleast one non-naturally occurring nucleotide or at least onenon-naturally occurring internucleotide linkage.
 16. The syntheticantisense molecule of claim 15, which is a synthetic microRNA (miRNA), asynthetic pri-miRNA, or a synthetic pre-miRNA.
 17. The syntheticantisense molecule of claim 15, wherein the synthetic antisense moleculecomprises the sequence of SEQ ID NO:
 179. 18. A recombinant expressionvector comprising a nucleotide sequence encoding an antisense moleculewhich specifically binds to a portion of an IRES of a CACNA1A genecomprising the sequence of SEQ ID NO:
 180. 19. (canceled)
 20. Therecombinant expression vector of claim 18, wherein the antisensemolecule comprises the sequence of SEQ ID NO: 179 or SEQ ID NO: 181.21-22. (canceled)
 23. The recombinant expression vector of claim 18,which is an recombinant adeno-associated viral (AAV) vector. 24-25.(canceled)
 26. The recombinant expression vector of claim 23, whereinthe recombinant AAV vector comprises one or more of a humancytomegalovirus (CMV) immediate early promoter, a pair of AAV ITRs, asimian virus 40 (SV40) polyadenylation signal sequence, a woodchuckhepatitis virus posttranscriptional regulatory element (WPRE), or acombination thereof. 27-29. (canceled)
 30. A cell comprising (i) anantisense molecule which specifically binds to a portion of an IRES of aCACNA1A gene comprising the sequence of SEQ ID NO: 180, (ii) therecombinant expression vector of any one of claims 18-28, (iii) anextracellular vesicle of claim 29, or (iv) a combination thereof. 31-32.(canceled)
 33. A pharmaceutical composition comprising (i) an antisensemolecule, (ii) a vector encoding the antisense molecule, (iii) a cellcomprising the vector or antisense molecule, (iv) a extracellularvesicle comprising the antisense molecule, or (v) a combination thereof,wherein the antisense molecule binds to a portion of an IRES of aCACNA1A gene comprising the base sequence of SEQ ID NO: 180 or SEQ IDNO: 179, and a pharmaceutically acceptable carrier, diluent, orexcipient, wherein the pharmaceutically acceptable carrier, diluent, orexcipient is synthetic, when the antisense molecule, cell, orextracellular vesicle is naturally occurring. 34-39. (canceled)
 40. Akit comprising (i) an antisense molecule, (ii) a vector encoding theantisense molecule, (iii) a cell comprising the vector or antisensemolecule, (iv) a extracellular vesicle comprising the antisensemolecule, or (v) a combination thereof, wherein the antisense moleculebinds to a portion of an IRES of a CACNA1A gene comprising the basesequence of SEQ ID NO: 180 or SEQ ID NO: 179, (ii) and a device foradministration to a subject. 41-46. (canceled)
 47. The method of claim 1wherein the antisense molecule binds to Argonaute 4 (Ago4).
 48. Thesynthetic antisense molecule of claim 15 wherein the synthetic antisensemolecule binds to Argonaute 4 (Ago4).
 49. The recombinant expressionvector of claim 18 wherein the recombinant expression vector comprises anucleotide sequence encoding an antisense molecule that binds toArgonaute 4 (Ago4).
 50. The cell of claim 30, wherein the cell comprisesan antisense molecule that binds to Argonaute 4 (Ago4).
 51. Thepharmaceutical composition of claim 33, wherein the pharmaceuticalcomposition comprises an antisense molecule that binds to Argonaute 4(Ago4).
 52. The kit of claim 40, wherein the kit comprises an antisensemolecule that binds to Argonaute 4.